WO2024086776A1 - Hybrid microbead arrays - Google Patents
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- WO2024086776A1 WO2024086776A1 PCT/US2023/077391 US2023077391W WO2024086776A1 WO 2024086776 A1 WO2024086776 A1 WO 2024086776A1 US 2023077391 W US2023077391 W US 2023077391W WO 2024086776 A1 WO2024086776 A1 WO 2024086776A1
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Classifications
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
- C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
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- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/251—Colorimeters; Construction thereof
- G01N21/253—Colorimeters; Construction thereof for batch operation, i.e. multisample apparatus
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
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- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
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Definitions
- Biomolecules are composed of diverse cells, extracellular matrix, and complex signaling systems organized to interact and execute complex functions. It has been well established that tissue functions essentially rely on the precise spatial organization of cells characterized by distinct molecular profiles. In particular, cells within a tissue of a subject have differences in cell morphology and/or function due to varied levels of biomolecules and metabolites within the different cells. Therefore, determining the spatial distribution of biomolecules can be of great significance for life sciences research, molecular diagnostics and many other applications. In addition to understanding the gene expression profile of a particular cell or tissue, spatial information of biomolecules (e.g., nucleic acids, chemical metabolites, proteins, etc.) within the cell or tissue may also provide valuable information.
- biomolecules e.g., nucleic acids, chemical metabolites, proteins, etc.
- Some spatial transcriptomics platforms involve the transfer and capture of cellular analytes, or proxies thereof, from a tissue section to barcoded capture probes.
- the capture probes are generally immobilized on a substrate or feature.
- Features may take a variety of forms, one of which is a bead.
- Bead microarrays are widely used in biological analyses and multiplexing Attorney Docket No.: 057862-516001WO applications.
- Most bead arrays are unsuitable for imaging with a biological sample (e.g., tissue section) on top, due to the refraction created by the beads.
- the disclosure relates to methods of preparing an array including: (a) providing a substrate comprising a plurality of features attached to a surface of the substrate, wherein a feature of the plurality of features has a first refractive index at a first wavelength, (b) providing a filling material, wherein the filling material has a second refractive index at the first wavelength, and wherein the difference between the first refractive index and the second refractive index is no greater than about 10%; and (c) applying the filling material to the surface of the substrate such that spaces between the plurality of features attached to the surface of the substrate substantially comprise the filling material.
- the methods further include applying a force to the filling material on the surface of the substrate and/or to the substrate such that the filling material is substantially uniformly distributed across the surface of the substrate.
- the force is a centrifugal or mechanical force.
- the methods further include subjecting the filling material on the surface of the substrate to desiccation, wherein an amount of trapped gas in the filling material following desiccation is less than an amount of trapped gas in the filling material prior to desiccation.
- the substrate includes a plurality of microwells, wherein a microwell of the plurality of microwells includes a feature.
- the feature is attached to a surface of the microwell.
- the plurality of microwells is etched on the surface of the substrate.
- the plurality of microwells is electroplated on the surface of the substrate.
- the plurality of microwells is photolithographically deposited on the surface of the substrate.
- the surface of the substrate is functionalized with one or more silanes, amines, or a combination thereof.
- the substrate is transparent and/or translucent. In some embodiments, the substrate is glass. In some embodiments, the substrate is silica. [0011] In some embodiments of the methods of preparing the arrays of the disclosure, the filling material includes a polymer, optionally wherein the polymer is in the form of a gel, a hydrogel, or a viscous liquid. In some embodiments, the polymer is polydimethylsiloxane (PDMS). [0012] In some embodiments of the methods of preparing the arrays of the disclosure, the filling material is reversibly attached to the substrate.
- PDMS polydimethylsiloxane
- the filling material forms a layer on the surface of the substrate comprising a thickness in the range of about 500 nanometers to about 50 micrometers.
- the filling material is in a fluid state when applied to the surface of the substrate, and the methods further include solidifying or polymerizing the filling material, e.g., via heating, UV curing, or cross-linking.
- the feature is attached to the substrate via heating, gluing, UV curing, or cross-linking.
- the feature is attached to the substrate via electrostatic interactions or mechanical fixation.
- the feature is a bead.
- the bead has a diameter of about 0.1 ⁇ m to about 5 ⁇ m, about 1 ⁇ m to about 10 ⁇ m, about 1 ⁇ m to about 20 ⁇ m, about 1 ⁇ m to about 30 ⁇ m, about 1 ⁇ m to about 40 ⁇ m, about 1 ⁇ m to about 50 ⁇ m, about 1 ⁇ m to about 60 ⁇ m, about 1 ⁇ m to about 70 ⁇ m, about 1 ⁇ m to about 80 ⁇ m, about 1 ⁇ m to about 90 ⁇ m, about 90 ⁇ m to about 100 ⁇ m, about 80 ⁇ m to about 100 ⁇ m, about 70 ⁇ m to about 100 ⁇ m, about 60 ⁇ m to about 100 ⁇ m, Attorney Docket No.: 057862-516001WO about 50 ⁇ m to about 100 ⁇ m, about 40 ⁇ m to about 100 ⁇ m, about 30 ⁇ m, about 1 ⁇ m to about 40 ⁇ m, about 1 ⁇ m to about 50
- the feature is composed of a material selected from the group consisting of silica, polystyrene, hydrogel, and a combination thereof.
- the filling material and the composition of the feature are substantially the same, and/or the feature and the filling material have the same refractive index at the first wavelength. In some embodiments, the filling material and the composition of the feature are different.
- the feature has a location on the substrate and the feature includes a plurality of polynucleotide capture probes, such that a polynucleotide capture probe in the plurality of polynucleotide capture probes includes a bead barcode and a capture domain, wherein the capture domain is capable of binding to an analyte.
- every polynucleotide capture probe of the plurality of polynucleotide capture probes on the feature includes the same bead barcode.
- the plurality of polynucleotide capture probes on the feature includes a different bead barcode than other polynucleotide capture probes on other features of the plurality of features.
- the analyte is mRNA or DNA.
- the capture domain includes a poly(T) sequence.
- the bead barcode of the feature is associated with the location of the feature on the substrate.
- the methods further include determining the sequence of the bead barcode.
- the determining includes sequencing.
- the sequencing is in situ sequencing.
- in situ sequencing is performed via sequencing-by-synthesis (SBS), sequential fluorescence hybridization, sequencing by ligation, nucleic acid hybridization, or high-throughput digital sequencing techniques.
- SBS sequencing-by-synthesis
- the disclosure provides arrays prepared by the methods of the disclosure.
- the disclosure provides methods for detecting a biological analyte in a biological sample including: (a) providing an array having a plurality of features attached to a surface of a substrate prepared by any of the methods of the disclosure, (b) contacting the biological sample with the array, (c) imaging the biological sample to generate an image of the biological sample, (d) incubating the biological sample under conditions wherein the biological analyte binds to a capture probe on a feature of the plurality of features, (e) determining a location of the analyte on the surface of the substrate; and (f) mapping the location of the analyte on the surface of the substrate onto a location in the biological sample using the image of the biological sample.
- the determining a location of the analyte on the surface of the substrate includes sequencing.
- the sequencing is in situ sequencing.
- the in situ sequencing is performed via sequencing-by-synthesis (SBS), sequential fluorescence hybridization, sequencing by ligation, nucleic acid hybridization, or high- throughput digital sequencing techniques.
- a distortion in the image of the biological sample resulting from the feature is reduced as compared to a distortion in a reference image of the biological sample, wherein the reference image is generated using a corresponding array that does not include the filling material (e.g., an array having the same features and substrate, but not the filling material) during the imaging.
- the filling material e.g., an array having the same features and substrate, but not the filling material
- the disclosure provides methods for detecting a biological analyte in a biological sample including: (a) contacting the biological sample to an array of the disclosure, (b) imaging the biological sample to generate an image of the biological sample, (c) incubating the biological sample under conditions wherein the biological analyte binds a capture probe on a feature in the plurality of features, (d) determining (i) all or a part of the sequence of the biological analyte specifically bound to the capture probe, or a complement thereof, and (ii) the sequence of the bead barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to identify the location of the analyte on the surface of the substrate; and (e) Attorney Docket No.: 057862-516001WO mapping the location of the analyte on the surface of the substrate onto a location in the biological sample using the image of the biological sample.
- a distortion in the image of the biological sample resulting from the feature is reduced as compared to a distortion in a reference image of the biological sample, wherein the reference image is generated using a corresponding spatial array that does not comprise the filling material during the imaging.
- the disclosure provides methods for detecting a biological analyte in a biological sample including: (a) contacting the biological sample to an array prepared by any of the methods of the disclosure, (b) incubating the biological sample under conditions wherein the biological analyte binds a polynucleotide capture probe on a feature in the plurality of features, (c) determining (i) all or a part of the sequence of the biological analyte specifically bound to the polynucleotide capture probe, or a complement thereof, and (ii) the sequence of the bead barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to identify the location of the analyte on the surface of the substrate.
- the biological sample includes fresh tissue, frozen tissue, formalin fixed, or formalin- fixed, paraffin-embedded tissue.
- the methods further include attaching the biological sample to the array and/or permeabilizing the biological sample to release the biological analyte therefrom, optionally wherein the permeabilizing includes the use of an organic solvent, a detergent, an enzyme, or a combination thereof.
- the biological analyte is a nucleic acid.
- the nucleic acid is selected from the group consisting of mRNA, gDNA, rRNA, and tRNA.
- the methods for detecting a biological analyte of the disclosure further include fixing the biological sample.
- the fixing the biological sample includes use of a fixative selected from the group consisting of: ethanol, methanol, acetone, formaldehyde, paraformaldehyde-Triton, glutaraldehyde, and combinations thereof.
- the methods further include staining the biological sample, imaging the biological sample, or a combination thereof.
- the staining includes the use of eosin and/or hematoxylin.
- the staining includes use of a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.
- the feature is a bead.
- the disclosure provides arrays including: (a) a substrate having a plurality of features attached to the substrate, wherein a feature of the plurality of features has a first refractive index at a first wavelength; and (b) a filling material, wherein the filling material has a second refractive index at the first wavelength, and wherein the difference between the first refractive index and the second refractive index is no greater than about 10%, wherein the filling material is disposed on a surface of the substrate where the plurality of features are attached, and wherein the filling material substantially fills the spaces between the plurality of features attached to the surface of the substrate.
- the substrate further includes a plurality of microwells, and wherein a microwell of the plurality of microwells comprises a feature of the plurality of features.
- the feature is attached to a surface of the microwell.
- the plurality of microwells is etched on the surface of the substrate.
- the plurality of microwells is electroplated on the surface of the substrate.
- the plurality of microwells is photolithographically deposited on the surface of the substrate.
- the surface of the substrate is functionalized with one or more silanes, amines, or a combination thereof.
- the substrate is transparent and/or translucent.
- the substrate is glass. In some embodiments, the substrate is silica. [0036] In some embodiments of the arrays of the disclosure, the filling material comprises a polymer, optionally wherein the polymer is in the form of a gel, a hydrogel, or a viscous liquid. In some embodiments, the polymer is polydimethylsiloxane (PDMS). In some embodiments, the filling material is reversibly attached to the substrate. In some embodiments, the filling material Attorney Docket No.: 057862-516001WO forms a layer on the surface of the substrate comprising a thickness in the range of about 500 nanometers to about 50 micrometers.
- PDMS polydimethylsiloxane
- the filling material is in a fluid state when applied to the surface of the substrate, and/or the method further comprises solidifying or polymerizing the filling material via heating, UV curing, or cross-linking.
- the feature is attached to the substrate via heating, gluing, UV curing, or cross-linking. In some embodiments, the feature is attached to the substrate via electrostatic interactions or mechanical fixation. [0038] In some embodiments of the arrays, the feature is a bead.
- the bead has a diameter of about 0.1 ⁇ m to about 5 ⁇ m, about 1 ⁇ m to about 10 ⁇ m, about 1 ⁇ m to about 20 ⁇ m, about 1 ⁇ m to about 30 ⁇ m, about 1 ⁇ m to about 40 ⁇ m, about 1 ⁇ m to about 50 ⁇ m, about 1 ⁇ m to about 60 ⁇ m, about 1 ⁇ m to about 70 ⁇ m, about 1 ⁇ m to about 80 ⁇ m, about 1 ⁇ m to about 90 ⁇ m, about 90 ⁇ m to about 100 ⁇ m, about 80 ⁇ m to about 100 ⁇ m, about 70 ⁇ m to about 100 ⁇ m, about 60 ⁇ m to about 100 ⁇ m, about 50 ⁇ m to about 100 ⁇ m, about 40 ⁇ m to about 100 ⁇ m, about 30 ⁇ m to about 100 ⁇ m, about 20 ⁇ m to about 100 ⁇ m, or about 10 ⁇ m to about 100 ⁇ m.
- the feature is composed of a material selected from the group consisting of silica, polystyrene, hydrogel, and a combination thereof.
- the filling material and the composition of the feature are substantially the same, and/or the feature and the filling material have the same refractive index at the first wavelength. In some embodiments, the filling material and the composition of the feature are different.
- the feature has a location on the substrate and includes a plurality of polynucleotide capture probes, wherein a polynucleotide capture probe in the plurality of polynucleotide capture probes includes a bead barcode and a capture domain, such that the capture domain is capable of binding to an analyte.
- every polynucleotide capture probe of the plurality of polynucleotide capture probes on the feature includes the same bead barcode.
- the plurality of polynucleotide capture probes on the feature of the plurality of features includes a Attorney Docket No.: 057862-516001WO different bead barcode than other polynucleotide capture probes on other features of the plurality of features.
- the analyte is mRNA or DNA.
- the capture domain includes a poly(T) sequence optionally wherein the poly(T) sequence comprises about 20-30 thymidine nucleotides.
- the bead barcode is associated with the specific location of the feature on the substrate.
- the disclosure provides kits for spatial analysis of a biological analyte in a biological sample including any of the arrays of the disclosure.
- the kit optionally further includes instructions for use thereof.
- the biological analyte is a nucleic acid.
- the nucleic acid is selected from the group consisting of messenger RNA (mRNA), genomic DNA (gDNA), ribosomal RNA (rRNA), and transfer RNA (tRNA).
- kits further include one or more fixatives for fixing the biological sample.
- the fixative is selected from the group consisting of: ethanol, methanol, acetone, formaldehyde, paraformaldehyde-Triton, glutaraldehyde, and combinations thereof.
- the kits further include one or more staining reagents for staining the biological sample.
- the staining reagents include eosin and/or hematoxylin.
- the one or more staining reagents include a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.
- a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.
- FIG.1 schematically illustrates an exemplary embodiment of the hybrid microbead arrays of the present disclosure.
- the substrate can be, but is not necessarily, transparent or translucent.
- FIG.2 schematically illustrates another exemplary embodiment of the hybrid microbead arrays with microwells of the present disclosure.
- FIG.3 schematically shows steps for an exemplary embodiment of a method of preparing a hybrid microbead array of the present disclosure.
- FIG.4 shows the optical transparency of a 5 ⁇ m silica bead array (FIG.4A) and a polydimethylsiloxane (PDMS) filler material deposited on the 5 ⁇ m silica bead array (FIG.4B).
- PDMS polydimethylsiloxane
- the disclosure relates to compositions and methods for improving tissue-imaging quality of bead arrays used in such platforms. More particularly, the present disclosure provides arrays (bead arrays) and methods of preparing arrays that include a filling material. The present disclosure also provides methods for detecting biological analytes, or proxies thereof, using the arrays of the disclosure, as well as kits including said arrays.
- Spatial transcriptomic systems may involve the transfer and capture of analytes (e.g., cellular transcripts) from biological samples (e.g. tissue sections) onto bead arrays.
- the present disclosure provides, inter alia, bead arrays composed of beads embedded in a filling material.
- the bead arrays of the disclosure include beads and filling material such that the difference between the refractive index of the beads and the refractive index of the filling material is not greater than about 10%. Matching the index of refraction of the beads and the filling material reduces refraction of light passing through the biological sample, such as during imaging for example.
- the bead arrays of the disclosure have several advantages over bead arrays that do not have a filling material.
- the bead arrays of the disclosure have better adhesion and retention of beads on the substrate, and have improved optical transparency and/or less optical distortion.
- the bead arrays of the disclosure provide an improved surface for the capture of cellular transcripts and other analytes from biological tissues. Furthermore, the filling material enhances the robustness of the bead arrays by providing support for the beads from vibration and other physical interactions, such as etching or scratches on the bead array surface. D EFINITIONS [0056] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
- nucleic acids are capable of hybridizing to a nucleic acid in a sequence-specific fashion (e.g., capable of hybridizing to two nucleic acids such that ligation can occur between the two hybridized nucleic acids) or are capable of being used as a template for replication of a particular nucleotide sequence.
- Naturally-occurring nucleic acids generally have a backbone containing phosphodiester bonds.
- An analog structure can have an alternate backbone linkage including any of a variety of those known in the art.
- Naturally-occurring nucleic acids generally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid (DNA)) or a ribose sugar (e.g., found in ribonucleic acid (RNA)).
- a nucleic acid can contain nucleotides Attorney Docket No.: 057862-516001WO having any of a variety of analogs of these sugar moieties that are known in the art.
- a nucleic acid can include native or non-native nucleotides.
- a native deoxyribonucleic acid can have one or more bases selected from the group consisting of adenine (A), thymine (T), cytosine (C), or guanine (G), and a ribonucleic acid can have one or more bases selected from the group consisting of uracil (U), adenine (A), cytosine (C), or guanine (G).
- uracil U
- A adenine
- C cytosine
- G guanine
- Useful non-native bases that can be included in a nucleic acid or nucleotide are known in the art.
- polynucleotide refers to a single-stranded multimer of nucleotides which can be from about 2 to about 500 nucleotides in length.
- Polynucleotides can be synthetic, made enzymatically (e.g., via polymerization), or using a “split-pool” method.
- Polynucleotides can include ribonucleotide monomers (i.e., can be oligoribonucleotides) and/or deoxyribonucleotide monomers (i.e., oligodeoxyribonucleotides).
- polynucleotides can include a combination of both deoxyribonucleotide monomers and ribonucleotide monomers in the polynucleotide (e.g., random or ordered combination of deoxyribonucleotide monomers and ribonucleotide monomers).
- a polynucleotide can be 4 to 10, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 500 nucleotides or more in length, for example.
- Polynucleotides can include one or more functional moieties that are attached (e.g., covalently or non-covalently) to the multimer structure.
- a polynucleotide can include one or more detectable labels (e.g., a radioisotope or fluorophore).
- detectable labels e.g., a radioisotope or fluorophore.
- barcode is used herein to refer to a label, or identifier, that conveys or is capable of conveying information (e.g., information about an analyte in a sample, a bead, and/or a nucleic acid barcode molecule).
- a barcode can be part of an analyte or nucleic acid barcode molecule, or independent of an analyte or nucleic acid barcode molecule.
- a barcode can be attached to an analyte or nucleic acid barcode molecule in a reversible or irreversible manner.
- a particular barcode can be unique relative to other barcodes. Barcodes can have a variety of different formats.
- barcodes may include polynucleotide barcodes, random nucleic Attorney Docket No.: 057862-516001WO acid and/or amino acid sequences, and synthetic nucleic acid and/or amino acid sequences.
- a barcode can be attached to an analyte or to another moiety or structure in a reversible or irreversible manner.
- a barcode can be added to, for example, a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before or during sequencing of the sample. Barcodes can allow for or facilitate identification and/or quantification of individual sequencing- reads.
- a barcode can be configured for use as a fluorescent barcode.
- a barcode can be configured for hybridization to fluorescently labeled oligonucleotide capture probes. Barcodes may be configured to spatially resolve molecular components found in biological samples, for example, at single-cell resolution (e.g., a barcode can be or can include a “spatial barcode”).
- a barcode includes two or more sub-barcodes that together function as a single barcode.
- a polynucleotide barcode may include two or more polynucleotide sequences (e.g., sub-barcodes). In some embodiments, the two or more sub-barcodes are separated by one or more non-barcode sequences.
- the two or more sub-barcodes are not separated by non- barcode sequences.
- the term “cell” refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell, cell culture, or cell line, without regard to the number of transfers or passages in culture. It should be understood that not all progeny are exactly identical to the parental cell.
- the term “attach,” and grammatical derivatives thereof, refer to affixing, securing, associating, fastening, adhering, bonding, or connecting by a bond, link, force (e.g., magnetic force), or tie in order to keep two or more elements together.
- attach encompasses either direct or indirect attachments, and also encompasses both covalent attachments and noncovalent attachments.
- the term includes but is not limited to fixing, securing, associating, fastening, or Attorney Docket No.: 057862-516001WO connecting the feature directly or indirectly to the substrate.
- the term “susbtantially” is generally used to indicate approximation and refers to “largely but not necessarily wholly that which is specified.”
- the term“substantially fills,” when used with respect to filling spaces between the features attached to the surface of the substrate, generally refers to applying a volume of the filling material into the spaces between the features such that the volume fills (e.g., occupies) substantially all of the spaces, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or any value in between, of the spaces between the features attached to the surface.
- the filling material occupies from 80% to 99%, from 85% to 98%, from 90% to 97%, or from 95% to 96% of the spaces between the features attached to the surface of the substrate.
- substantially fills when used to refer to a particular space (i.e. one individual space), it generally refers to applying a volume of the filling material into substantially all the particular space, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or any value in between, of the one individual space.
- the filling material occupies from 80% to 99%, from 85% to 98%, from 90% to 97%, or from 95% to 96% of the one individual space.
- Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.
- compositions are synonymous with “including”, “containing”, or “characterized by”, and is inclusive or open-ended and does not exclude additional, unrecited elements or method Attorney Docket No.: 057862-516001WO steps.
- consisting of excludes any elements, steps, or ingredients not specified in the claimed composition or method.
- consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term “comprising”, particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or steps.
- the present disclosure provides, inter alia, arrays for use in spatial transcriptomic analysis.
- the arrays of the disclosure can include (a) a substrate having a plurality of features attached to the substrate, and (b) a filling material such that the filling material substantially fills the spaces between the plurality of features.
- the features have a first refractive index at a first wavelength (e.g., 1.45) and the filling material has a second refractive index (e.g., 1.4) at the first wavelength.
- the first refractive index and the second refractive index are the same.
- the refractive index can be a dimensionless value representing the relative ratio of the speed of light in a medium (e.g., a filling material) relative to the speed of light in a vacuum.
- Refractive index can be used to describe the phase velocity of light through a medium, e.g., how much the path of light is refracted when entering a medium (e.g., a filling material).
- the refractive index of a sample is dependent on temperature and wavelength.
- Refractive index can be measured using known methods in the art such as, for example, with an Abbe refractometer, an ellipsometer, or a prism coupler refractometer.
- the first refractive index and the second refractive index are different at the same (e.g., a first) wavelength.
- the first wavelength is a wavelength of visible light.
- the first wavelength is in the range of about 380 to about 700 nm, or a subrange therein.
- the difference between the first refractive index and the second refractive index is no greater than about 10%.
- the difference is no greater than about 1%, no greater than 1.5%, no greater than about 2%, no greater than about 2.5%, no greater than about 3%, no greater than about 3.5%, no greater than about 4%, no greater than about 4.5%, no greater than about 5%, no greater than about 5.5%, no greater than about 6%, no greater than about 6.5%, no greater than about 7%, no greater than about 7.5%, no greater than about 8%, no greater than about 8.5%, no greater than about 9%, no greater than about 9.5%, no greater than about 10% or any values in between.
- the features have a first refractive index of about 1.4, about Attorney Docket No.: 057862-516001WO 1.41, about 1.42, about 1.43, about 1.44, about 1.45, about 1.46, about 1.47, about 1.48, or about 1.49 or within any range having any two of these values as endpoints.
- the filling material has a second refractive index about 1.4, about 1.41, about 1.42, about 1.43, about 1.44, about 1.45, about 1.46, about 1.47, about 1.48, or about 1.49 or within any range having any two of these values as endpoints.
- the filling material of the arrays of the disclosure can be made from various types of material.
- the filling material can be made of one or more polymers.
- the polymer can be in the form of gels, hydrogels, or viscous liquids.
- the polymer is polydimethylsiloxane (PDMS), polyethylene, polypropylene, PET, PMMA, or a combination thereof.
- PDMS polydimethylsiloxane
- the filling material of the arrays of the disclosure can have a thickness in the range of about 500 nanometers to about 50 micrometers when disposed on the substrate. In some embodiments, the thickness of the filling material on the surface of the substrate is in the range of about 400 nanometers to about 45 micrometers.
- the thickness of the filling material on the surface of the substrate is in the range of about 300 nanometers to about 40 micrometers, or in the range of about 200 nanometers to about 35 micrometers, or in the range of about 100 nanometers to about 30 micrometers.
- the thickness of the filling material on the surface of the substrate can correspond to the size, volume, circumference and/or diameter of the beads of the array. In one embodiment, the thickness of the filling material can correspond to an amount of filling material needed to substantially cover or envelope the beads of the array.
- the filling material of the disclosure can be in a fluid state when applied to the surface of the substrate. In some embodiments, the filling material is solidified after application to the substrate.
- the filling material is polymerized after application to the substrate. In some embodiments, the filling material is gelled after application to the substrate.
- the filling material can be gelled, solidified or polymerized by any suitable method known in the art. In some embodiments, the filling material is solidified or polymerized by heating. In some embodiments, the filling material is solidified or polymerized by UV curing. In some embodiments, the filling material is solidified or polymerized by cross-linking. In some embodiments, the filling material is solidified or polymerized by adding an initiator, adding Attorney Docket No.: 057862-516001WO shear, or changing the pH.
- the filling material covers or substantially covers all spaces in between the features. In some embodiments, the filling material covers the top of the features on the array as shown, for example, in FIG.1 or FIG 2. In some embodiments, the filling material partially covers the features of the array. [0079]
- the filling material can be permanently attached to the substrate or can be reversibly attached to the substrate. In some embodiments, the filling material is removed when needed. In some embodiments, the filling material is dissolved and removed from the array after certain processes. [0080] In some embodiments, the filling material is subjected to desiccation in order to remove or reduce the formation of air bubbles during the filling process.
- the filling material is subjected to a negative pressure (e.g., vacuum) to remove or reduce formation of air bubbles during the filling process.
- a negative pressure e.g., vacuum
- a specific arrangement of a plurality of features can be either irregular or can form a regular pattern. Individual features on the array can differ from one another based on their relative spatial locations.
- a plurality of the features of the disclosure are spatially ordered in an array and each feature of the plurality of the features is associated with a unique spatial location on the array.
- the arrays of the disclosure are designed to accommodate a large number of features.
- the array can include at least 5,000 features, for example at least 10,000 features, at least 20,000 features, at least 50,000 features, at least 100,000 features, at least 500,000 features, at least 1,000,000 features, at least 2,000,000 features, at least 5,000,000 features, or at least 10,000,000 features, or more.
- the plurality of features includes a single type of feature (e.g., substantially uniform in volume, shape, and other physical properties, such as translucence). In some embodiments, the plurality of features includes two or more types of different features.
- the arrays of the disclosure can include arrays prepared by any of the methods of the disclosure discussed below.
- the arrays described herein include features which are designed and/or configured to act as supports or repositories for various molecular entities or proxies thereof used in tissue sample analysis.
- the features may be formulated into various shapes and dimensions depending on the context of intended use. Examples of features include, but are not limited to, a bead, a spot of any two- or three-dimensional geometry (e.g., an ink jet spot, a masked spot, a square on a grid), a microwell, and a hydrogel pad. In some preferred embodiments, the features are beads.
- the features are microbeads (i.e., beads having a largest dimension of less than one thousand micrometers).
- the feature is a microbead.
- the microbead may have an average diameter of about 0.1 micrometer (micron) to about 50 micrometers.
- the microbead may have an average diameter of about 0.1 micrometer (micron) to about 100 micrometers.
- the microbead may have an average diameter of about 0.1 micrometer, about 0.2 micrometers, about 0.3 micrometers, about 0.4 micrometers, about 0.5 micrometers, about 0.6 micrometers, about 0.7 micrometers, about 0.8 micrometers, or about 0.9 micrometers.
- the microbead may have an average diameter of about 1 micron, about 2 micrometers, about 3 micrometers, about 4 micrometers, about 5 micrometers, about 6 micrometers, about 7 micrometers, about 8 micrometers, about 9 micrometers, about 10 micrometers, about 11 micrometers, about 12 micrometers, about 13 micrometers, about 14 micrometers, about 15 micrometers, about 16 micrometers, about 17 micrometers, about 18 micrometers, about 19 micrometers, about 20 micrometers, about 25 micrometers, about 30 micrometers, about 35 micrometers, about 40 micrometers, about 45 micrometers, about 50 micrometers, about 60 micrometers, about 70 micrometers, about 80 micrometers, about 90 micrometers, about 100 micrometers or any other values in between.
- the bead (e.g., microbead) may have a diameter of about 0.1 micrometer to about 5 micrometers, about 1 micrometer to about 10 micrometers, about 1 micrometer to about 20 micrometers, about 1 micrometer to about 30 micrometers, about 1 micrometer to about 40 micrometers, about 1 micrometer to about 50 micrometers, about 1 micrometer to about 60 micrometers, about 1 micrometer to about 70 micrometers about 1 Attorney Docket No.: 057862-516001WO micrometer to about 80 micrometers about 1 micrometer to about 90 micrometers about 1 micrometer to about 100 micrometers or any other ranges in between.
- the bead (e.g., microbead) has a diameter of about 0.1 ⁇ m, about 0.5 ⁇ m, about 1 ⁇ m, about 2 ⁇ m, about 3 ⁇ m, about 4 ⁇ m, about 5 ⁇ m, about 6 ⁇ m, about 7 ⁇ m, about 8 ⁇ m, about 9 ⁇ m, about 10 ⁇ m, about 11 ⁇ m, or about 12 ⁇ m.
- the features of the disclosure can be in the form of a polymer.
- the polymer can be in the form of a hydrogel, a gel, a viscous liquid or other forms.
- the features of the disclosure can be composed of various materials.
- Non-limiting examples of materials include silica, polystyrene, carboxyl-modified polystyrene, polystyrene/2% divinylbenzene, polystyrene/10% divinylbenzene, polystyrene/55% divinylbenzene, polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), or any combination thereof.
- the features are composed of silica, polystyrene, hydrogel or a combination thereof.
- the feature may be further modified by silane deposition and/or polymer adsorption.
- the features e.g., beads
- the features are optically transparent or translucent.
- the features are optically transparent or translucent when the substrate is imaged at a first wavelength.
- the features of the arrays of the disclosure can be attached to a substrate via a variety of methods. Non-limiting examples of how features can be attached to the substrate include chemical bonding (e.g., via a covalent bond), electrostatic interactions, mechanical fixation, or other methods. In some embodiments, the features can be attached to the substrate via heating, gluing, UV curing, cross-linking or by functionalization e.g., via salines or amines, or by other methods known to those skilled in the art.
- the features of the disclosure can be amine modified and arrayed with EDC-coupling on N-hydroxysuccinimide glass slides.
- the features are attached to the top surface of the substrate.
- the features are located partially or completely within microwells in the substrate.
- each microwell in the substrate includes a single feature.
- some or all the features of the disclosure are functionalized Attorney Docket No.: 057862-516001WO for analyte capture.
- the features may include capture probes (including e.g., capture domains) for analyte capture.
- the term capture probe generally refers to any molecule capable of capturing (directly or indirectly) and/or labelling an analyte of interest in or from a biological sample.
- the capture probes may include a nucleic acid binding moiety (NABM), which can be small molecules, aptamers, proteins, or oligonucleotides.
- NABM nucleic acid binding moiety
- the capture probes are polynucleotide capture probes.
- the capture probes may include ribonucleotides and/or deoxyribonucleotides as well as synthetic nucleotide residues that are capable of participating in Watson-Crick type or analogous base pair interactions.
- the capture probes may include a conjugate (e.g., an oligonucleotide-antibody conjugate).
- a feature can occupy a discrete location on the substrate and include a plurality of capture probes (e.g., polynucleotide capture probes) such that the capture probes on the feature include a bead barcode and a capture domain capable of binding or hybridizing to an analyte or a proxy thereof.
- the features may include a plurality of capture probes at an average density of about 500 to about 100,000 capture probes per micrometer 2 . In some embodiments, the average density is about 1,000 to about 100,000 capture probes per micrometer 2 .
- the average density is about 10,000 to about 80,000 capture probes per micrometer 2 . In some embodiments, the average density is about 25,000 to about 60,000 capture probes per micrometer 2 .
- the features may include a plurality of capture probes at an average density of at least about 500 molecules per ⁇ m 2 , at least about 1000 molecules per ⁇ m 2 , at least about 1500 molecules per ⁇ m 2 , at least about 2000 molecules per ⁇ m 2 , at least about 5000 molecules per ⁇ m 2 , at least about 10,000 molecules per ⁇ m 2 , at least about 15,000 molecules per ⁇ m 2 , at least about 20,000 molecules per ⁇ m 2 , at least about 25,000 molecules per ⁇ m 2 , at least about 30,000 molecules per ⁇ m 2 , at least about 35,000 molecules per ⁇ m 2 , at least about 40,000 molecules per ⁇ m 2 , at least about 45,000 molecules per ⁇ m 2 , at least about 50,000 molecules per ⁇ m 2 , at least about
- Capture probes may include a plurality of capture probes.
- the capture probes of the disclosure may be directly or indirectly attached, incorporated, embedded, and/or affixed to the features.
- the feature is a hydrogel having a plurality of moieties and the capture probe is attached to a moiety of the plurality of moieties within the hydrogel.
- the capture probe may include a barcode sequence (e.g., a spatial barcode, a bead barcode) and/or a capture domain.
- the bead barcode can be specific to a single bead so that different beads have different bead barcodes.
- the capture probes of the disclosure may include capture domains.
- the capture domain may be capable of binding (e.g., hybridizing to) an analyte or a proxy thereof.
- the capture domain may include a nucleic acid sequence (e.g., a poly(T) sequence) capable of binding to a poly(A) tail of an mRNA and/or to a poly(A) homopolymeric sequence present in genomic DNA.
- a homopolymeric sequence is added to an mRNA molecule or a genomic DNA molecule using a terminal transferase enzyme in order to produce a molecule that has a poly(A) or poly(T) sequence.
- a poly(A) sequence can be added to an analyte (e.g., a fragment of genomic DNA) thereby making the analyte capable of capture by a poly(T) capture domain.
- a capture domain includes a poly(T) sequence, a random sequence, a gene specific sequence, a degenerate sequence, or a combination thereof.
- the random sequence includes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more random nucleotides.
- the random sequence includes a random hexamer. In some embodiments, the random sequence includes a random decamer.
- the capture probe may include a unique molecular identifier (UMI). UMIs can be used for determining the number of unique molecules in a nucleic acid Attorney Docket No.: 057862-516001WO library (e.g., a NGS library) as described below in more detail.
- Each capture probe can optionally include at least one cleavage domain. The cleavage domain represents a portion of the capture probe that is used to reversibly attach the capture probe to a feature.
- one or more segments or regions of the capture probe can optionally be released from the feature by cleavage of the cleavage domain.
- spatial (e.g., bead) barcodes and/or universal molecular identifiers (UMIs) can be released by cleavage of the cleavage domain.
- the capture probe may be optionally coupled to a feature by a cleavage domain, such as a disulfide linker.
- a cleavage domain is absent from the capture probe. Examples of substrates with attached capture probes lacking a cleavage domain are described for example in Macosko et al., (2015) Cell 161, 1202–1214.
- each capture probe of the disclosure may optionally include at least one, at least two, a least three, or at least four functional domains or regions.
- Each functional domain or region generally includes a functional nucleotide sequence for a downstream analytical step in the overall analysis procedure.
- a capture probe of the disclosure can include (i) a sequencing domain, (ii) a PCR adaptor, (iii) a primer binding site and/or (iv) a capture region capable of binding to a target analyte or a proxy thereof.
- the capture probe can include adapters that are useful for subsequent downstream processing.
- the spatial barcode (e.g., bead barcodes) of the disclosure includes a contiguous nucleic acid segment or at least two non-contiguous nucleic acid segments that function as a label or identifier that conveys or is capable of conveying spatial information.
- a capture probe can include a spatial barcode (e.g., spatial bead barcode) that possesses a spatial aspect, where the bead barcode is associated with a particular location within an array or a particular location on a substrate.
- a bead barcode can be a spatial barcode once the bead is positioned on the substrate and associated with a unique position thereon.
- a spatial barcode can be incorporated within the capture probe for use in barcoding a target analyte Attorney Docket No.: 057862-516001WO or a proxy thereof.
- the functional sequences of a barcode can generally be designed or selected for compatibility with any of a variety of different sequencing systems, e.g., 454 Sequencing, Ion Torrent Proton or PGM, Illumina sequencing instruments, PacBio, Nanopore, etc., and the requirements thereof. In some embodiments, functional sequences can be designed or selected for compatibility with non-commercialized sequencing systems.
- a spatial barcode may be part of an analyte, or independent from an analyte (e.g., part of the capture probe).
- a spatial barcode can be a tag attached to an analyte (e.g., a nucleic acid molecule) or a combination of a tag in addition to an endogenous characteristic of the analyte (e.g., size of the analyte).
- the spatial barcode can be common to all of the capture probes attached to a given feature (feature barcode).
- a spatial barcode can be unique.
- the spatial barcode functions both as a spatial barcode and as a unique molecular identifier (UMI), associated with one particular capture probe.
- UMI unique molecular identifier
- the spatial barcode and UMI are separate nucleic acid sequences and their functions within a capture probe do not overlap.
- Spatial barcodes can have a variety of different formats.
- spatial barcodes can include polynucleotide spatial barcodes, random nucleic acid and/or amino acid sequences, and synthetic nucleic acid and/or amino acid sequences.
- a spatial barcode is attached to an analyte in a reversible or irreversible manner.
- a spatial barcode is added to, for example, a fragment of a DNA or RNA sample before, during, and/or after downstream analyses (e.g., sequencing) of the sample.
- a spatial barcode is designed or selected to allow for identification and/or quantification of individual sequencing-reads.
- a spatial barcode is used as a fluorescent barcode for which fluorescently labeled oligonucleotide capture probes hybridize and/or attach to the spatial barcode.
- the spatial barcode is a nucleic acid sequence that does not substantially hybridize to analyte nucleic acid molecules in a biological sample.
- the spatial barcode has less than 80% sequence identity (e.g., less than 70%, 60%, 50%, or less than 40% sequence identity) to the nucleic acid sequences across a substantial part (e.g., 80% or more) of the nucleic acid molecules in the biological sample.
- 80% sequence identity e.g., less than 70%, 60%, 50%, or less than 40% sequence identity
- the spatial barcode sequences of the multiple capture probes can include sequences that are the same for all capture probes coupled to the feature, and/or sequences that are different across all capture probes coupled to the feature.
- capture probes attached to a single feature can include identical (or common) spatial barcode sequences, different spatial barcode sequences, or a combination of both.
- Capture probes attached to a feature can include multiple sets of capture probes.
- Capture probes of a given set can include identical spatial barcode sequences.
- the identical spatial barcode sequences can be different from spatial barcode sequences of capture probes of another set.
- the identical spatial barcode sequences can be different from spatial barcode sequences of capture probes attached to another feature.
- the plurality of capture probes can include spatial barcode sequences (e.g., nucleic acid barcode sequences) that are associated with specific locations on a spatial array.
- a first plurality of capture probes can be associated with a first region, based on a spatial barcode sequence common to the capture probes within the first region, and a second plurality of capture probes can be associated with a second region, based on a spatial barcode sequence common to the capture probes within the second region.
- the second region may or may not be associated with the first region.
- Additional pluralities of capture probes can be associated with spatial barcode sequences common to the capture probes within other regions.
- the spatial barcode sequences can be the same across a plurality of capture probe molecules. [0116] In some embodiments, multiple different spatial barcodes are incorporated into a single feature.
- a capture probe of the disclosure can include at least one capture domain.
- the “capture domain” can be an oligonucleotide, a polypeptide, a small molecule, or any Attorney Docket No.: 057862-516001WO combination thereof, that binds specifically (e.g., hybridizes) to a desired analyte or class of analytes, or a proxy thereof.
- a capture domain can be used to directly or indirectly capture or detect a desired analyte.
- the capture domain is a functional nucleic acid sequence configured to interact with one or more analytes or proxies thereof, such as one or more different types of nucleic acids (e.g., RNA molecules and DNA molecules).
- the functional nucleic acid sequence can include an N-mer sequence (e.g., a random N-mer sequence), which N-mer sequences are configured to interact with a plurality of DNA molecules.
- the functional sequence can include a poly(T) sequence.
- poly(T) sequences are configured to interact with messenger RNA (mRNA) molecules via the poly(A) tail of an mRNA transcript.
- poly(T) sequences are configured to interact with a target nucleic acid (e.g., RTL probe) via a poly(A) sequence contained within the target nucleic acid.
- the functional nucleic acid sequence is the binding target of a protein (e.g., a transcription factor, a DNA binding protein, or a RNA binding protein), where the analyte of interest is a protein.
- the capture domain is capable of priming a reverse transcription reaction to generate cDNA that is complementary to captured RNA molecules.
- the capture domain of the capture probe can prime a DNA extension (polymerase) reaction to generate DNA that is complementary to the captured DNA molecules.
- the capture domain can template a ligation reaction between captured DNA molecules and a surface capture probe that is directly or indirectly immobilized on the substrate.
- the capture domain can be ligated to one strand of the captured DNA molecules.
- SplintR ligase along with RNA or DNA sequences (e.g., degenerate RNA) can be used to ligate a single-stranded DNA or RNA to the capture domain.
- ligases with RNA-templated ligase activity can be used to ligate a single-stranded DNA or RNA to the capture domain.
- a capture domain includes a splint oligonucleotide sequence.
- a capture domain captures a splint oligonucleotide.
- the capture domain is located at the 3’ end of the capture probe and includes a free 3’ end that can be extended, e.g., by template dependent Attorney Docket No.: 057862-516001WO polymerization, to form an extended capture probe as described herein.
- the capture domain includes a nucleotide sequence that is capable of hybridizing to nucleic acid, e.g., RNA or other analyte, present in the cells of the biological sample contacted with the array.
- the capture domain can be selected or designed to bind selectively or specifically to a target nucleic acid.
- the capture domain can be selected or designed to capture mRNA by way of hybridization to the mRNA poly(A) tail.
- the capture domain includes a poly(T) DNA oligonucleotide, e.g., a series of consecutive deoxythymidine residues linked by phosphodiester bonds, which is capable of hybridizing to the poly(A) tail of mRNA.
- the capture domain can include nucleotides that are functionally or structurally analogous to a poly(T) tail.
- a poly(U) oligonucleotide or an oligonucleotide included of deoxythymidine analogues are examples of deoxythymidine analogues.
- the capture domain includes at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides (e.g., T or U). In some embodiments, the capture domain includes at least 25, 30, or 35 nucleotides (e.g., T or U).
- random sequences e.g., random hexamers, decamers, or similar sequences, can be used to form all or a part of the capture domain. For example, random sequences can be used in conjunction with poly(T) (or poly(T) analogue) sequences.
- a capture domain includes a poly(T) (or a “poly(T)-like”) oligonucleotide
- it can also include a random oligonucleotide sequence (e.g., “poly(T)-random sequence” capture probe). This can, for example, be located 5’ or 3’ of the poly(T) sequence, e.g., at the 3’ end of the capture domain.
- the poly(T)-random sequence capture probe can facilitate capture of the mRNA poly(A) tail.
- the capture domain can be an entirely random sequence.
- degenerate capture domains can be used.
- the capture domain can be based on a particular gene sequence or particular motif sequence or common/conserved sequence, that it is designed to capture (i.e., a sequence-specific capture domain).
- the capture domain can bind (e.g., is configured for binding) selectively to a desired sub-type or subset of nucleic acid, for example a particular type of RNA, such as mRNA, rRNA, tRNA, SRP RNA, tmRNA, snRNA, snoRNA, SmY RNA, scaRNA, gRNA, RNase P, Rnase MRP, TERC, SL RNA, aRNA, cis-NAT, crRNA, lncRNA, miRNA, piRNA, siRNA, shRNA, tasiRNA, rasiRNA, 7SK, eRNA, ncRNA or other types of Attorney Docket No.: 057862-516001WO RNA.
- the capture domain can be capable of, or configured for, binding selectively to a desired subset of ribonucleic acids, for example, microbiome RNA, such as 16S rRNA.
- a capture domain includes an “anchor” or “anchoring sequence”, which is a sequence of nucleotides that is designed to ensure that the capture domain hybridizes to the intended analyte, or proxy thereof.
- an anchor sequence includes a sequence of nucleotides, including a 1-mer, 2-mer, 3-mer or longer sequence.
- the sequence is random.
- a capture domain including a poly(T) sequence can be designed to capture an mRNA.
- an anchoring sequence can include a random 3-mer (e.g., GGG) that helps ensure that the poly(T) capture domain hybridizes to an mRNA.
- an anchoring sequence can be VN, N, or NN.
- the sequence can be designed using a specific sequence of nucleotides.
- the anchor sequence is at the 3’ end of the capture domain. In some embodiments, the anchor sequence is at the 5’ end of the capture domain. [0124]
- capture domains of capture probes are blocked prior to contacting the biological sample with the array, and blocking probes may be used when the nucleic acids in the biological sample are modified prior to capture on the array.
- the blocking probe is used to block or modify the free 3’ end of the capture domain.
- blocking probes can be hybridized to the capture probes to mask the free 3’ end of the capture domain, e.g., hairpin probes, partially double stranded probes, or complementary sequences.
- the free 3’ end of the capture domain can be blocked by chemical modification, e.g., addition of an azidomethyl group as a chemically reversible capping moiety such that the capture probes do not include a free 3’ end.
- 3’ modifications include dideoxy C-3’ (3’-ddC), 3’ inverted dT, 3’ C3 spacer, 3’Amino, and 3’ phosphorylation.
- the nucleic acid in the biological sample can be modified such that it can be captured by the capture domain.
- an adaptor sequence (including a binding domain capable of binding to the capture Attorney Docket No.: 057862-516001WO domain of the capture probe) can be added to the end of the target nucleic acid, e.g., fragmented genomic DNA. In some embodiments, this is achieved by ligation of the adaptor sequence or extension of the nucleic acid. In some embodiments, an enzyme is used to incorporate additional nucleotides at the end of the nucleic acid sequence, e.g., a poly(A) tail. In some embodiments, the capture probes can be reversibly masked or modified such that the capture domain of the capture probe does not include a free 3’ end.
- the 3’ end is removed, modified, or made inaccessible so that the capture domain is not susceptible to the process used to modify the nucleic acid of the biological sample, e.g., ligation or extension.
- the capture domain of the capture probe is modified to allow the removal of any modifications of the capture probe that occur during modification of the nucleic acid molecules of the biological sample.
- the capture probes can include an additional sequence downstream of the capture domain, e.g., 3’ to the capture domain, namely a blocking domain.
- the capture domain of the capture probe can be a non- nucleic acid domain.
- Suitable capture domains that are not exclusively nucleic-acid based include, but are not limited to, proteins, peptides, aptamers, antigens, antibodies, and molecular analogs that mimic the functionality of any of the capture domains described herein.
- Unique molecular Identifiers UMI
- the capture probes of the disclosure can include one or more (e.g., two or more, three or more, four or more, five or more) Unique Molecular Identifiers (UMIs).
- a unique molecular identifier is a contiguous nucleic acid segment or two or more non-contiguous nucleic acid segments that function as a label or identifier for a particular analyte, or for a capture probe that binds a particular analyte or a proxy thereof (e.g., via the capture domain).
- a UMI can include one or more specific polynucleotides sequences, one or more random nucleic acid and/or amino acid sequences, and/or one or more synthetic nucleic acid and/or amino acid sequences.
- the UMI is a nucleic acid sequence that does not substantially hybridize to analyte nucleic acid molecules in a biological sample.
- the UMI has less than 80% sequence identity (e.g., less than 70%, 60%, 50%, or less than 40% sequence identity) to the nucleic acid sequences across a substantial part (e.g., Attorney Docket No.: 057862-516001WO 80% or more) of the nucleic acid molecules in the biological sample.
- sequence identity e.g., less than 70%, 60%, 50%, or less than 40% sequence identity
- These nucleotides can be completely contiguous, i.e., in a single stretch of adjacent nucleotides, or they can be separated into two or more separate subsequences that are separated by one or more nucleotides.
- a UMI is attached to an analyte in a reversible or irreversible manner.
- a UMI is added to, for example, a fragment of a DNA or RNA sample before or during, sequencing of the analyte.
- a UMI allows for identification and/or quantification of individual sequencing-reads.
- a UMI is a used as a fluorescent barcode for which fluorescently labeled oligonucleotide probes hybridize to the UMI. Functionally, UMIs allow for demultiplexing of sequencing reads and biases that may be introduced through downstream processing (e.g., PCR amplification).
- each biomolecule captured by a capture probe on a feature of a spatial bead array should contain a distinct UMI that is distinguishable from other UMIs on the spatial bead array.
- a substrate may be designed and/or configured to function as a support for direct or indirect attachment of the features of the disclosure.
- Features may be directly or indirectly attached, embedded, or affixed to a substrate.
- the features are not directly or indirectly attached embedded, or affixed to a substrate, but instead, for example, are disposed within an enclosed or partially enclosed three- dimensional space (e.g., wells, microwells or divots).
- the substrate includes a plurality of microwells.
- a microwell in the plurality of microwells include a feature.
- the microwells are etched on the surface of the substrate.
- the microwells are electroplated on the surface of the substrate.
- the microwells are photolithographically deposited on the surface of the substrate.
- the features of the disclosure are arrayed with EDC- coupling on N-hydroxysuccinimide glass slides.
- the surface of the substrate is functionalized with silanes, Attorney Docket No.: 057862-516001WO amines, and/or other suitable moieties.
- Substrates of the disclosure can be formed from a variety of solid materials, gel- based materials, colloidal materials, semi-solid materials (e.g., materials that are at least partially cross-linked), materials that are fully or partially cured (e.g., via photolithography), and materials that undergo a phase change or transition to provide physical support. Substrates of the disclosure can be insoluble in aqueous liquid.
- substrates of the disclosure include, but are not limited to, slides (e.g., slides formed from various glasses, slides formed from various polymers), layers and/or films, membranes (e.g., porous membranes), flow cells, cuvettes, wafers (e.g., silicon), plates, or combinations thereof.
- substrates can optionally include functional elements such as recesses, protruding structures, microfluidic elements (e.g., channels, reservoirs, electrodes, valves, seals), and various markings such a fiducials.
- a substrate can generally have any suitable form or format.
- a substrate can be flat, curved, e.g., convexly or concavely curved towards the area where the interaction between the substrate and a biological sample, e.g., tissue sample, takes place.
- a substrate is flat, e.g., planar, chip, or slide.
- a substrate can contain one or more patterned surfaces within the substrate (e.g., channels, wells, projections, ridges, divots, hydrophobically defined spaces intra hydrophillically defined spaces, etc.).
- Substrates of the disclosure can be of any desired shape.
- a substrate can be generally a thin, flat shape (e.g., a square or a rectangle).
- a substrate structure has rounded corners (e.g., for increased safety or robustness). In some embodiments, a substrate structure has one or more cut-off corners (e.g., for use with a slide clamp or cross-table). In some embodiments, where a substrate structure is flat, the substrate structure can be any appropriate type of support having a flat surface (e.g., a chip or a slide such as a microscope slide). [0141] In some embodiments of the disclosure, substrates can optionally include various structures such as, but not limited to, projections, ridges, and channels.
- a substrate can be micro- patterned to limit lateral diffusion (e.g., to prevent overlap of spatial barcodes or barcoded Attorney Docket No.: 057862-516001WO features).
- a substrate including such structures can be modified to allow association of analytes, features (e.g., beads), or capture probes at individual sites or regions.
- the sites or regions where a substrate is modified with various structures can be contiguous or non- contiguous with other sites or regions.
- the surface of a substrate can be modified so that discrete sites or regions are formed that can only have or accommodate a specific number of features, for example, a single feature.
- the surface of a substrate can be modified so that features adhere to random sites or regions.
- the surface of a substrate can be modified so that features are substantially regularly spaced from one another on the substrate.
- the surface of a substrate is modified to contain one or more wells, using techniques such as (but not limited to) stamping, microetching, photlithography, or molding techniques.
- the substrate in which a substrate includes one or more wells, can be a concavity slide or cavity slide.
- wells can be formed by one or more shallow depressions on the surface of the substrate.
- the wells can be formed by attaching a cassette (e.g., a cassette containing one or more chambers) to a surface of the substrate structure.
- the structures can include physically altered sites or regions.
- a substrate modified with various structures can include physical properties, including, but not limited to, physical configurations, magnetic or compressive forces, chemically functionalized sites, chemically altered sites, and/or electrostatically altered sites.
- the structures are applied in a pattern.
- the structures can be randomly distributed.
- the structures can be regularly spaced from one another on the substrate.
- a substrate can be any suitable support material.
- Exemplary Attorney Docket No.: 057862-516001WO substrates include, but are not limited to, glass, modified and/or functionalized glass, films, membranes, plastics (e.g., acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon TM , cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, and polymers, such as polystyrene, cyclic olefin copolymers (COCs), cyclic olef
- the substrates can include glass, silicon dioxide or a silicon wafer. In some embodiments, the substrates are made of glass. In some embodiments, the substrates are made of quartz or silica. In some embodiments, the substrates are transparent. In some embodiments, the substrates are translucent. [0146] In some embodiments, the substrate is a conductive substrate. Conductive substrates (e.g., electrophoretic compatible arrays) generated as described herein can be used in the spatial detection of analytes or proxies thereof.
- Conductive substrates e.g., electrophoretic compatible arrays
- an electrophoretic field can be applied to facilitate migration of analytes towards the barcoded features (e.g., features immobilized on a wafer, features or immobilized in a hydrogel film, or features immobilized on a glass slide having a conductive coating).
- a conductive substrate can include glass (e.g., a glass slide) that has been coated with a substance or otherwise modified to confer conductive properties to the glass.
- kits are for the spatial analysis of a nucleic acid analyte (e.g., mRNA, gDNA, rRNA, or tRNA).
- a kit can include one or more substrates including the features and/or filling materials of the disclosure.
- a kit can further include instructions for using the components of the kit to practice a method described herein.
- the components of a kit can be in separate containers. In some other embodiments, the components of a kit can be combined in a single container. In some embodiments, the kit includes fixatives for fixing the biological sample.
- the fixatives are ethanol, methanol, acetone, formaldehyde, paraformaldehyde, Triton, glutaraldehyde, or combinations thereof.
- the kit includes staining reagents for staining the biological sample (e.g., eosin and/or hematoxylin).
- the kit includes a detectable label for use in staining the biological sample (e.g., a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof).
- the instructions for practicing the method are generally recorded on a suitable recording medium.
- the instructions can be printed on a substrate, such as paper or plastic, etc.
- the instructions can be present in the kit as a package insert, in the labeling of the container of the kit or components thereof (e.g., associated with the packaging or sub- packaging), etc.
- the instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, flash drive, etc.
- the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet), can be provided.
- An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.
- M ETHODS OF THE DISCLOSURE Methods for Preparing Arrays [0150] The disclosure provides, inter alia, methods for preparing an array (e.g., hybrid microbead array), the methods include providing a substrate with a plurality of features attached to the substrate, providing a filling material, and applying the filling material to the surface of the substrate such that spaces between the plurality of features attached to the surface of the substrate substantially comprise the filling material.
- the disclosure provides methods for preparing an array, the methods include providing a substrate comprising a plurality of features attached to the substrate, wherein a feature of the plurality of features has a first refractive index at a first wavelength, providing a filling material, wherein the filling material has a second refractive index at the first wavelength, and wherein the difference between the first refractive index and the second refractive index is Attorney Docket No.: 057862-516001WO no greater than about 10%, and applying the filling material to the surface of the substrate such that spaces between the plurality of features attached to the surface of the substrate comprise the filling material.
- the first wavelength is a wavelength of visible light.
- the first wavelength is in the range of about 380 to about 700 nm, or a subrange therein.
- a feature of the plurality of features can have a first refractive index at a first wavelength and the filling material can have a second refractive index at the first wavelength, such that the difference between the first refractive index and the second refractive index is no greater than about 10%.
- the first refractive index and the second refractive index are different at the same wavelength.
- the difference between the first refractive index and the second refractive index is no greater than about 10%.
- the difference is no greater than about 1%, no greater than about 1.5%, no greater than about 2%, no greater than about 2.5%, no greater than about 3%, no greater than about 3.5%, no greater than about 4%, no greater than about 4.5%, no greater than about 5%, no greater than about 5.5%, no greater than about 6%, no greater than about 6.5%, no greater than about 7%, no greater than about 7.5%, no greater than about 8%, no greater than about 8.5%, no greater than about 9%, no greater than about 9.5%, no greater than about 10% or any values in between.
- the first refractive index and the second refractive index are the same.
- the fillings can be specifically selected to match the index of refraction of the features so as to reduce the refraction of light passing through the biological sample (e.g., tissue section).
- the methods of preparing an array of the disclosure can include a further step of applying a force to the filling material on the surface of the substrate and/or to the substrate such that the filling material is substantially uniformly distributed across the surface of the substrate. Any form of a suitable force can be used as appreciated by the skilled artisan.
- the force is a mechanical force.
- the force is a centrifugal force.
- the centrifugal force is generated by spinning the substrate.
- the methods of preparing an array of the disclosure can include a further step of subjecting the filling material on the surface of the substrate to desiccation, such that any trapped gas in the filling material is reduced or eliminated. Any methods for releasing trapped gas in the filling may be used. For example, subjecting the filling material on the surface of the substrate to shaking or vibration can be used to release trapped gas.
- the methods of preparing an array of the disclosure include sequencing a bead barcode within a capture probe of a feature, e.g., in order to determine the location of an analyte or a proxy thereof.
- the sequence of the bead barcode can be associated with the location of the bead (e.g., feature) on the substrate.
- the sequencing is in situ sequencing.
- the in situ sequencing is performed via sequencing-by-synthesis (SBS), sequential fluorescence hybridization, sequencing by ligation (SBL), nucleic acid hybridization, or high-throughput digital sequencing techniques.
- SBS sequencing-by-synthesis
- SBL sequencing by ligation
- nucleic acid hybridization or high-throughput digital sequencing techniques.
- the disclosure provides methods for detecting a biological analyte in a biological sample, the methods include: (a) providing an array of the disclosure; (b) contacting the biological sample to the array, (c) imaging the biological sample to generate an image of the biological sample, (d) incubating the biological sample under conditions wherein the biological analyte binds a capture probe on a feature of the array, I determining a location of the analyte on the surface of the substrate, and (f) mapping the location of the analyte on the surface of the substrate onto a location in the biological sample using the image of the biological sample.
- the disclosure provides methods for detecting a biological analyte in a biological sample, the methods include: (a) contacting the biological sample to an array prepared by the methods of the disclosure, (b) imaging the biological sample to generate an image of the biological sample, (c) incubating the biological sample under conditions wherein the biological analyte binds a capture probe on a feature in the plurality of features of the arrays, Attorney Docket No.: 057862-516001WO (d) determining (i) all or a part of the sequence of the biological analyte specifically bound to the capture probe, or a complement thereof, and (ii) the sequence of the bead barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to identify the location of the analyte on the surface of the substrate, aI(e) mapping the location of the analyte on the surface of the substrate onto a location in the biological sample using the image of the biological sample.
- the disclosure provides methods for detecting a biological analyte in a biological sample, the methods include (a) contacting the biological sample to an array prepared by the methods described herein, (b) incubating the biological sample under conditions wherein the biological analyte binds a capture probe on a feature in the plurality of features, (c) determining (i) all or a part of the sequence of the biological analyte specifically bound to the capture probe, or a complement thereof, and (ii) the sequence of the bead barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to identify the location of the analyte on the surface of the substrate.
- the methods of detecting a biological analyte of the disclosure can include sequencing a bead barcode on a capture probe of a feature in order to determine the location of an analyte or a proxy thereof.
- the sequence of the bead barcode can be associated with the specific location of the bead (or feature) on the substrate.
- the sequencing is in situ sequencing.
- the in situ sequencing is performed via sequencing-by-synthesis (SBS), sequential fluorescence hybridization, sequencing by ligation (SBL), nucleic acid hybridization, or high-throughput digital sequencing techniques.
- the methods of detecting a biological analyte of the disclosure can further include disposing or attaching the biological sample to the substrate and/or permeabilizing the biological sample to release the biological analyte therefrom.
- the permeabilizing can include the use of an organic solvent, a detergent, an enzyme, or a combination thereof.
- a distortion in the image of the biological sample resulting from the feature is reduced as compared to a distortion in a reference image of the biological sample.
- a reference image is generated using a corresponding array that does not include the filling Attorney Docket No.: 057862-516001WO material, in particular during the imaging step.
- the biological sample can be a tissue sample, such as, fresh tissue, frozen tissue, formalin-fixed, or formalin-fixed and paraffin embedded tissue (e.g., FFPE tissue).
- the biological sample is a tissue section, such as, a fresh and/or frozen tissue section or an FFPE tissue section.
- the biological analyte can be a nucleic acid, a mRNA, a gDNA, a rRNA, and/or a tRNA.
- the methods further include fixing the biological sample.
- the fixing is by using a fixative.
- the fixative can be any fixative including, but not limited to ethanol, methanol, acetone, formaldehyde, paraformaldehyde-Triton, glutaraldehyde, and combinations thereof.
- the methods for detecting a biological analyte of the disclosure include staining the biological sample.
- Staining can be performed by using a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound or any combination thereof. In some embodiments, staining is performed by using eosin and/or hematoxylin.
- Spatial Analysis An exemplary workflow for the array-based spatial analysis methods disclosed herein involves the transfer of one or more analytes, or proxies thereof, from a biological sample to an array of the disclosure where a feature in the array is associated with a unique spatial location on the array.
- the analyte e.g., a nucleic acid
- proxy thereof hybridizes to a capture domain on the array.
- Subsequent analysis of the transferred analytes includes determining the identity of the analytes and the spatial location of each analyte within the biological sample.
- the spatial location of each analyte within the biological sample is determined based on the feature to which each analyte is bound or hybridized to on the array, and the feature’s relative spatial location within the array.
- the biological analytes are proteins, in which case a set of analyte capture agents (e.g., antibody-oligonucleotide conjugates) or nucleic acid capture probes are applied to the biological sample, and the capture probes bind to the oligonucleotide of the antibody-oligonucleotide conjugates or the ligated capture probe set following hybridization and/or binding to a target of interest (e.g., protein or nucleic acids).
- a target of interest e.g., protein or nucleic acids.
- the oligonucleotide can be cleaved from the antibody prior to being captured by the capture probe.
- the spatial analysis of a biological analyte present in a biological sample may begin, for example, with imaging a fresh or frozen tissue section for histological purposes and placing the tissue section on an array containing capture probes that bind to one or more biological analytes in the sample.
- the tissue section is fixed and permeabilized to release analytes which can be captured by capture probes disposed adjacent to the cells of the tissue section that the analytes are released from, which allows for or facilitates downstream analytical application of the captured analytes (e.g., spatially determined gene expression or protein activity).
- the captured nucleic acid or a nucleic acid derived therefrom is used for preparation of sequencing libraries.
- the libraries are sequenced, and sequencing data can be visualized, for example, to determine which genes are expressed, and where, as well as optionally, in what quantity, within a cell or tissue sample.
- the cDNA generated from mRNA captured by capture probes disposed adjacent to a specific spot or region of the tissue sample generally share a common spatial barcode.
- Sequencing libraries are then generated from the cDNA and sequenced and the spatial barcodes are subsequently used to associate the sequence reads back to the tissue section images for spatial gene expression analysis and mapping.
- Another example of a workflow can include preparing a biological sample on a spatially-barcoded array.
- Sample preparation may include placing the sample on a slide, fixing the sample, and/or staining the biological sample for imaging.
- the stained sample can be imaged on the array using brightfield (to image the sample with, for example, a hematoxylin and eosin stain) and/or fluorescence (to image features comprising fluorescent moieties) modalities in such a way that positions in the spatially-barcoded array can be mapped to positions in the sample.
- the sample can be destained prior to permeabilization.
- analytes are released from the sample and capture probes forming the spatially-barcoded array hybridize to the released analytes.
- the analyte-bound capture probes can be analyzed to determine the identity of the analyte and where it was located on the spatial array.
- the Attorney Docket No.: 057862-516001WO analyte is RNA (e.g., mRNA)
- the RNA can be reverse transcribed into cDNA containing information from the spatial barcode of the capture probe hybridized to the RNA, and an amplicon sequencing library can be prepared and sequenced to identify the RNA and where it was located on the spatial array.
- RNA transcripts present in biological samples can be used for spatial transcriptome analysis.
- the barcoded oligonucleotides may be configured to prime, replicate, and consequently yield barcoded extension products from an RNA template, or derivatives thereof.
- the barcoded oligonucleotides may include mRNA specific priming sequences, e.g., poly-T primer segments that allow priming and replication of mRNA in a reverse transcription reaction or other targeted priming sequences.
- random RNA priming may be carried out using random N-mer primer segments of the barcoded oligonucleotides.
- Reverse transcriptases can use an RNA template and a primer complementary to the 3’ end of the RNA template to direct the synthesis of the first strand complementary DNA (cDNA).
- cDNA first strand complementary DNA
- Many RTs can be used in this reverse transcription reactions, including, for example, avian myeloblastosis virus (AMV) reverse transcriptase, moloney murine leukemia virus (M-MuLV or MMLV), and other variants thereof.
- AMV avian myeloblastosis virus
- M-MuLV or MMLV moloney murine leukemia virus
- Some recombinant M-MuLV reverse transcriptases can have reduced RNase H activity and increased thermostability when compared to their wild type counterparts, and provide higher specificity, higher yield of cDNA and more full-length cDNA products with up to 12 kilobase (kb) in length.
- the reverse transcriptase enzyme is a mutant reverse transcriptase enzyme such as, but not limited to, mutant MMLV reverse transcriptase.
- the reverse transcriptase is a mutant MMLV reverse transcriptase such as, but not limited to, one or more variants described in US Patent Publication No.20180312822.
- a biological sample e.g., tissue section
- methanol stained with hematoxylin and eosin
- imaged can be destained prior to permeabilization.
- the images can be Attorney Docket No.: 057862-516001WO used to map spatial gene expression patterns back to the biological sample.
- a permeabilization agent e.g., an enzyme
- Analytes e.g., polyadenylated mRNA
- released from the overlying cells of the biological sample can be captured by capture probes within a capture area on a substrate.
- Reverse transcription (RT) reagents can be added to the permeabilized biological samples. Incubation with the RT reagents can produce spatially-barcoded cDNA from the captured analytes (e.g., polyadenylated mRNA). Second strand reagents (e.g., second strand primers, enzymes) can be added to the biological sample on the substrate to initiate second strand synthesis. The resulting cDNA can be denatured from the capture probe template and transferred (e.g., to a clean tube) for amplification, and/or library construction. In some embodiments, the spatially-barcoded, full- length cDNA can be amplified via PCR prior to library construction.
- RT Reverse transcription
- the cDNA can be enzymatically fragmented and size-selected in order to optimize the cDNA amplicon size.
- P5, P7, i7, and i5 can be used as sample indexes, and TruSeq Read 2 can be added via End Repair, A-tailing, Adaptor Ligation, and PCR.
- the cDNA fragments can be sequenced using paired-end sequencing using TruSeq Read 1 and TruSeq Read 2 as sequencing primer sites (e.g., when utilizing Illumina based sequencers, other sequencing platforms may require different or additional sequencing primer sequences).
- performing correlative analysis of data produced by this workflow, and other workflows described herein can yield over 95% correlation of genes expressed across two capture areas (e.g., 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater).
- the cDNA can be amplified directly on the substrate surface. Generating multiple copies of the cDNA (e.g., cDNA synthesized from captured analytes) via amplification directly on the substrate surface can improve final sequencing library complexity.
- cDNA can be amplified directly on the substrate surface by isothermal nucleic acid amplification.
- isothermal nucleic acid amplification can amplify RNA or DNA.
- the staining Attorney Docket No.: 057862-516001WO includes optical labels as described herein, including, but not limited to, fluorescent, radioactive, chemiluminescent, calorimetric, or colorimetric detectable labels.
- the staining includes a fluorescent antibody directed to a target analyte (e.g., cell surface or intracellular proteins) in the biological sample.
- the staining includes an immunohistochemistry stain directed to a target analyte (e.g., cell surface or intracellular proteins) in the biological sample.
- the staining includes a chemical stain such as hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS).
- H&E hematoxylin and eosin
- PAS periodic acid-Schiff
- significant time e.g., days, months, or years
- reagents for performing analysis are added to the biological sample before, contemporaneously with, or after the array is contacted to the biological sample.
- the method may include placing the array onto the biological sample.
- the array is a flexible array where the plurality of spatially-barcoded features (e.g., with capture probes) are attached to a flexible substrate.
- measures are taken to slow down a reaction (e.g., cooling the temperature of the biological sample or using enzymes that preferentially perform their primary function at lower or higher temperature as compared to their optimal functional temperature) before the array is contacted with the biological sample.
- analyzing the analyte may be without bringing the biological sample out of contact with the array. In some embodiments, analyzing the analyte is performed after the biological sample is no longer in contact with the array.
- the biological sample is tagged with an analyte capture agent before, contemporaneously with, or after staining and/or imaging of the biological sample. In such cases, significant time (e.g., days, months, or years) can elapse between staining and/or imaging and performing analysis.
- the array is adapted to facilitate biological analyte migration from the stained and/or imaged biological sample onto the array (e.g., using any of the materials or methods described herein).
- a biological sample is permeabilized before being contacted with an array.
- the rate of permeabilization is slowed prior to contacting a biological sample with an array (e.g., to limit diffusion of analytes away from their original locations in the biological sample).
- modulating the rate of permeabilization e.g., modulating the activity of a permeabilization reagent
- modulating the rate of permeabilization can occur by modulating a condition that the biological sample is Attorney Docket No.: 057862-516001WO exposed to (e.g., modulating temperature, pH, and/or light).
- modulating the rate of permeabilization includes use of external stimuli (e.g., small molecules, enzymes, and/or activating reagents) to modulate the rate of permeabilization.
- a permeabilization reagent can be provided to a biological sample prior to contact with an array, which permeabilization reagent is inactive until a condition (e.g., temperature, pH, and/or light) is changed or an external stimulus (e.g., a small molecule, an enzyme, and/or an activating reagent) is provided.
- a condition e.g., temperature, pH, and/or light
- an external stimulus e.g., a small molecule, an enzyme, and/or an activating reagent
- Biological samples The methods of the disclosure, provided herein include detection and/or spatial analysis of a biological analyte in a biological sample.
- a biological sample is obtained from a subject for analysis using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and/or other biological material from the subject.
- the biological sample is a tissue sample e.g., comprising cells.
- the biological sample e.g., tissue sample
- the biological sample is a tissue microarray (TMA).
- TMA tissue microarray
- a tissue microarray contains multiple representative tissue samples – which can be from different tissues or organisms – assembled on a single histologic slide. The TMA can therefore allow for high throughput analysis of multiple specimens at the same time.
- Tissue microarrays are paraffin blocks produced by extracting cylindrical tissue cores from different paraffin donor blocks and re-embedding these into a single recipient (microarray) block at defined array coordinates.
- the tissue sample can be obtained from any suitable location in a tissue or organ of a subject, e.g., a human subject.
- the sample is a mouse sample. In some instances, the sample is a human sample. In some embodiments, the sample can be derived from skin, brain, breast, lung, liver, kidney, prostate, tonsil, thymus, testes, bone, lymph node, ovary, eye, heart, or spleen. [0177] In some embodiments, the tissue sample is a tissue section (e.g., tissue section obtained using a cryostat or microtome).
- a biological sample can be obtained from a mammal (e.g., a human), or non-mammalian organisms (e.g., a plants, an insect, an arachnid, a nematode (e.g., Caenorhabditis elegans), a fungi, an amphibian, or a fish (e.g., zebrafish)).
- a mammal e.g., a human
- non-mammalian organisms e.g., a plants, an insect, an arachnid, a nematode (e.g., Caenorhabditis elegans), a fungi, an amphibian, or a fish (e.g., zebrafish)
- a biological sample can be obtained from a prokaryote such as a bacterium, Attorney Docket No.: 057862-516001WO e.g., Escherichia coli, Staphylococci or Mycoplasma pneumoniae; an archaea; a virus such as Hepatitis C virus or human immunodeficiency virus; or a viroid.
- a biological sample can be obtained from a eukaryote, such as a patient derived organoid (PDO) or patient derived xenograft (PDX).
- PDO patient derived organoid
- PDX patient derived xenograft
- a biological sample can include organoids, a miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro-anatomy.
- Organoids can be generated from one or more cells from a tissue, embryonic stem cells, and/or induced pluripotent stem cells, which can self- organize in three-dimensional culture owing to their self-renewal and differentiation capacities.
- an organoid is a cerebral organoid, an intestinal organoid, a stomach organoid, a lingual organoid, a thyroid organoid, a thymic organoid, a testicular organoid, a hepatic organoid, a pancreatic organoid, an epithelial organoid, a lung organoid, a kidney organoid, a gastruloid, a cardiac organoid, or a retinal organoid.
- Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., cancer) or a pre-disposition to a disease, and/or individuals that are in need of therapy or suspected of needing therapy.
- Biological samples can include one or more diseased cells.
- a diseased cell can have altered metabolic properties, gene expression, protein expression, and/or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer.
- Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells.
- the biological sample e.g., the tissue sample
- a fixative including alcohol, for example methanol.
- alcohol for example methanol.
- acetone instead of methanol, acetone, or an acetone-methanol mixture can be used.
- the fixation is performed after sectioning.
- the biological sample e.g., the tissue sample
- the biological sample is fixed e.g., immediately after being harvested from a subject.
- the biological sample can be fixed using PAXgene.
- the biological sample can be fixed using PAXgene in addition, or alternatively to, a fixative disclosed herein or known in the art (e.g., alcohol, acetone, acetone-alcohol, formalin, paraformaldehyde).
- PAXgene is a non-cross-linking mixture of different alcohols, acid and a soluble organic compound that preserves morphology and biomolecules. It is a two-reagent fixative system in which tissue is firstly fixed in a solution Attorney Docket No.: 057862-516001WO containing methanol and acetic acid then stabilized in a solution containing ethanol. See, Ergin B.
- a biological sample can include a single analyte of interest, or more than one analyte of interest. Methods for performing multiplexed assays to analyze two or more different analytes in a single biological sample is discussed herein.
- the biological sample can be preserved after completion of an assay with a feature or arrangement of features for additional rounds of spatial detection of analytes.
- the biological sample, features, array, or any combination thereof can be preserved after the spatial profiling.
- the biological sample, features, array, or combinations thereof can be protected from dehydration (e.g., drying, desiccation).
- the biological sample, features, array, or combinations thereof can be protected from evaporation.
- reversible sealing agent Any suitable reversible sealing agent can be used. Methods of reversible sealing are known in the art (See, e.g., WO 2019/104337, which is incorporated herein by reference).
- suitable reversible sealing agents can include non-porous materials, membranes, lids, or oils (e.g., silicone oil, mineral oil).
- the biological sample, features, array, or combinations thereof can be preserved in an environmental chamber (e.g., hermetically sealed) and removed for additional rounds of spatial analysis at a later time.
- Biological Analytes [0183]
- the methods of the disclosure, provided herein include spatial analysis or detection of a biological analyte or proxy thereof in a biological sample. Attorney Docket No.: 057862-516001WO [0184]
- an analyte of the disclosure will be understood to include any biological substance, structure, moiety, or component to be analyzed.
- Analytes can be broadly classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes.
- non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral coat proteins, extracellular and intracellular proteins, antibodies, and antigen binding fragments.
- the analyte can be an organelle (e.g., nuclei or mitochondria).
- Cell surface features corresponding to analytes can include, but are not limited to, a receptor, an antigen, a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen-presenting complex, a major histocompatibility complex, an engineered T-cell receptor, a T-cell receptor, a B-cell receptor, a chimeric antigen receptor, an extracellular matrix protein, a posttranslational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation or lipidation) state of a cell surface protein, a gap junction, and an adherens junction.
- a posttranslational modification e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, ace
- Analytes can be derived from a specific type of cell and/or a specific sub-cellular region.
- analytes can be derived from cytosol, from cell nuclei, from mitochondria, from microsomes, and more generally, from any other compartment, organelle, or portion of a cell. Permeabilizing agents that specifically target certain cell compartments and organelles can be used to selectively release analytes from cells for analysis.
- nucleic acid analytes include DNA analytes such as genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and RNA/DNA hybrids.
- RNA analytes such as various types of coding and non-coding RNA.
- examples of the different types of RNA analytes include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), and viral RNA.
- the RNA can be a transcript (e.g., present in a tissue section).
- the Attorney Docket No.: 057862-516001WO RNA can be small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA greater than 200 nucleic acid bases in length).
- the RNA can be double-stranded RNA or single-stranded RNA.
- the RNA can be circular RNA.
- the RNA can be a bacterial rRNA (e.g., 16S rRNA or 23S rRNA).
- Additional examples of analytes include mRNA and cell surface features (e.g., using the labelling agents described herein), mRNA and intracellular proteins (e.g., transcription factors), mRNA and cell methylation status, mRNA and accessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq), mRNA and metabolites (e.g., using the labelling agents described herein), a barcoded labelling agent (e.g., the oligonucleotide tagged antibodies described herein) and a V(D)J sequence of an immune cell receptor (e.g., T-cell receptor), mRNA and a perturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc finger nuclease, and
- a perturbation agent can be a small molecule, an antibody, a drug, an aptamer, a miRNA, a physical environmental (e.g., temperature change), or any other known perturbation agents.
- Analytes can include a nucleic acid molecule with a nucleic acid sequence encoding at least a portion of a V(D)J sequence of an immune cell receptor (e.g., a TCR or BCR).
- the nucleic acid molecule is cDNA first generated from reverse transcription of the corresponding mRNA, e.g., using a poly(T) containing primer.
- the generated cDNA can then be barcoded using a capture probe, featuring a barcode sequence (and optionally, a UMI sequence) that hybridizes with at least a portion of the generated cDNA.
- a capture probe featuring a barcode sequence (and optionally, a UMI sequence) that hybridizes with at least a portion of the generated cDNA.
- Additional methods and compositions suitable for barcoding cDNA generated from mRNA transcripts including those encoding V(D)J regions of an immune cell receptor and/or barcoding methods and composition including a template switch oligonucleotide are described in PCT Patent Application PCT/US2017/057269, and PCT Patent Publication Nos. WO 2021/247568 and WO 2021/247543.
- V(D)J analysis can also be completed with the use of one or more labelling agents that bind to particular surface features of immune cells and associated with barcode sequences.
- the one or more labelling agents can include an MHC or MHC multimer.
- the analyte can include a nucleic acid capable of functioning as a component of a gene editing reaction, such as, for example, clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing.
- the capture probe can include a nucleic acid Attorney Docket No.: 057862-516001WO sequence that is complementary to the analyte (e.g., a sequence that can hybridize to the CRISPR RNA (crRNA), single guide RNA (sgRNA), or an adapter sequence engineered into a crRNA or sgRNA).
- micron functionalized microspheres were suspended in water at 1% solids w/v and 100 microliters of which were added to a well. The wells were sealed and allowed to settle at 4° C for several hours to overnight. The slide was removed from the cassette and gently rinsed by submersion in water until excess beads were removed leaving a monolayer. The slide was allowed to dry and then was placed onto a spin coater. A PDMS mixture was prepared and degassed by a vacuum desiccator for 10 min, and 2 mL PDMS was applied to the center of the slide and the slide was spun at 6000 rpm for 2 min. PDMS was cured at 100°C for 75 minutes and allowed to cool to room temperature.
- a method of preparing an array comprising: (a) providing a substrate comprising a plurality of features attached to a surface of the substrate, wherein a feature of the plurality of features has a first refractive index at a first wavelength; (b) providing a filling material, wherein the filling material has a second refractive index at the first wavelength, and wherein the difference between the first refractive index and the second refractive index is no greater than about 10%; and (c) applying the filling material to the surface of the substrate such that spaces between the plurality of features attached to the surface of the substrate substantially comprise the filling material.
- the substrate comprises a plurality of microwells, and wherein a microwell of the plurality of microwells comprises a feature. 6.
- the method of paragraph 5, wherein the feature is attached to a surface of the microwell. 7.
- the method of paragraph 5 or 6, wherein the plurality of microwells is etched on the surface of the substrate.
- the plurality of microwells is electroplated on the surface of the substrate.
- the plurality of microwells is photolithographically deposited on the surface of the substrate.
- the surface of the substrate is functionalized with one or more silanes, amines, or a combination thereof. 11.
- the substrate is transparent and/or translucent. 12. The method of paragraph 10, wherein the substrate is glass. 13. The method of paragraph 11, wherein the substrate is silica. Attorney Docket No.: 057862-516001WO 14. The method of any one of paragraphs 1-13, wherein the filling material comprises a polymer, optionally wherein the polymer is in the form of a gel, a hydrogel, or a viscous liquid. 15. The method of paragraph 14, wherein the polymer is polydimethylsiloxane (PDMS). 16. The method of any one of paragraphs 1-15, wherein the filling material is reversibly attached to the substrate. 17.
- any one of paragraphs 1-16 wherein the filling material forms a layer on the surface of the substrate comprising a thickness in the range of about 500 nanometers to about 50 micrometers. 18. The method of any one of paragraphs 1-17, wherein the filling material is in a fluid state when applied to the surface of the substrate, and wherein the method further comprises solidifying or polymerizing the filling material via heating, UV curing, or cross-linking. 19. The method of any one of paragraphs 1-18, wherein the feature is attached to the substrate via heating, gluing, UV curing, or cross-linking. 20. The method of any one of paragraphs 1-18, wherein the feature is attached to the substrate via electrostatic interactions or mechanical fixation. 21.
- the feature is a bead. 22.
- the bead has a diameter of about 0.1 ⁇ m to about 5 ⁇ m, about 1 ⁇ m to about 10 ⁇ m, about 1 ⁇ m to about 20 ⁇ m, about 1 ⁇ m to about 30 ⁇ m, about 1 ⁇ m to about 40 ⁇ m, about 1 ⁇ m to about 50 ⁇ m, about 1 ⁇ m to about 60 ⁇ m, about 1 ⁇ m to about 70 ⁇ m, about 1 ⁇ m to about 80 ⁇ m, about 1 ⁇ m to about 90 ⁇ m, about 90 ⁇ m to about 100 ⁇ m, about 80 ⁇ m to about 100 ⁇ m, about 70 ⁇ m to about 100 ⁇ m, about 60 ⁇ m to about 100 ⁇ m, about 50 ⁇ m to about 100 ⁇ m, about 40 ⁇ m to about 100 ⁇ m, about 30 ⁇ m to about 100 ⁇ m, Attorney Docket No.: 0578
- the feature has a location on the substrate and comprises a plurality of polynucleotide capture probes, wherein a polynucleotide capture probe in the plurality of polynucleotide capture probes comprises a bead barcode and a capture domain, and wherein the capture domain is capable of binding to an analyte.
- every polynucleotide capture probe of the plurality of polynucleotide capture probes on the feature comprises the same bead barcode.
- the method of paragraph 31, further comprising determining the sequence of the bead barcode.
- 33. The method of paragraph 32, wherein the determining comprises sequencing, and wherein the sequencing is in situ sequencing.
- the method of paragraph 33, wherein the in situ sequencing is performed via sequencing- by-synthesis (SBS), sequential fluorescence hybridization, sequencing by ligation, nucleic acid hybridization, or high-throughput digital sequencing techniques.
- 35. An array prepared by the method of any one of paragraphs 1-34. 36.
- a method for detecting a biological analyte in a biological sample comprising: (a) providing an array comprising a plurality of features attached to a surface of a substrate prepared by the method of any one of paragraphs 1-34; (b) contacting the biological sample with the array; (c) imaging the biological sample to generate an image of the biological sample; (d) incubating the biological sample under conditions wherein the biological analyte binds to a polynucleotide capture probe on a feature of the plurality of features; (e) determining a location of the analyte on the surface of the substrate; and (f) mapping the location of the analyte on the surface of the substrate onto a location in the biological sample using the image of the biological sample. 37.
- the method for detecting a biological analyte of paragraph 36, wherein determining a location of the analyte on the surface of the substrate comprises sequencing. 38.
- the method of any one of paragraphs 36 to 38, wherein the in situ sequencing is performed via sequencing-by-synthesis (SBS), sequential fluorescence hybridization, sequencing by ligation, nucleic acid hybridization, or high-throughput digital sequencing techniques. 40.
- SBS sequencing-by-synthesis
- a method for detecting a biological analyte in a biological sample comprising: (a) contacting the biological sample to an array prepared by the method of any one of paragraphs 26-34; (b) imaging the biological sample to generate an image of the biological sample; (c) incubating the biological sample under conditions wherein the biological analyte binds a polynucleotide capture probe on a feature in the plurality of features; (d) determining (i) all or a part of the sequence of the biological analyte specifically bound to the polynucleotide capture probe, or a complement thereof, and (ii) the sequence of the bead barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to identify the location of the analyte on the surface of the substrate; and (e) mapping the location of the analyte on the surface of the substrate onto a location in the biological sample using the image of the biological sample.
- a method for detecting a biological analyte in a biological sample comprising: Attorney Docket No.: 057862-516001WO (a) contacting the biological sample to an array prepared by the method of any one of paragraphs 26-34; (b) incubating the biological sample under conditions wherein the biological analyte binds a polynucleotide capture probe on a feature in the plurality of features; and (c) determining (i) all or a part of the sequence of the biological analyte specifically bound to the polynucleotide capture probe, or a complement thereof, and (ii) the sequence of the bead barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to identify the location of the analyte on the surface of the substrate.
- nucleic acid is selected from the group consisting of mRNA, gDNA, rRNA, and tRNA.
- the method further comprises fixing the biological sample.
- fixing the biological sample comprises use of a fixative selected from the group consisting of: ethanol, methanol, acetone, formaldehyde, paraformaldehyde-Triton, glutaraldehyde, and combinations thereof.
- Attorney Docket No.: 057862-516001WO 50 The method of any one of paragraphs 48 or 49, wherein the method further comprises staining the biological sample, imaging the biological sample, or a combination thereof. 51.
- the staining comprises use of eosin and/or hematoxylin.
- the staining comprises use of a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.
- 53. The method of any one of paragraphs 36 to 52, wherein the feature is a bead. 54.
- An array comprising: (a) a substrate comprising a plurality of features attached to the substrate, wherein a feature of the plurality of features has a first refractive index at a first wavelength; and (b) a filling material, wherein the filling material has a second refractive index at the first wavelength, and wherein the difference between the first refractive index and the second refractive index is no greater than about 10%, wherein the filling material substantially fills the spaces between the plurality of features attached to the surface of the substrate.
- the substrate further comprises a plurality of microwells, and wherein a microwell of the plurality of microwells comprises a feature of the plurality of features.
- the array of paragraph 55 wherein the feature is attached to a surface of the microwell. 57.
- the array of paragraph 55 or 56, wherein the plurality of microwells is etched on the surface of the substrate.
- the array of paragraphs 55 or 56, wherein the plurality of microwells is electroplated on the surface of the substrate.
- the array of paragraph 55 or 56, wherein the plurality of microwells is photolithographically deposited on the surface of the substrate.
- 60. The array of any one of paragraphs 54 to 59, wherein the surface of the substrate is functionalized with one or more silanes, amines, or a combination thereof. 61.
- PDMS polydimethylsiloxane
- any one of paragraphs 54 to 66 wherein the filling material forms a layer on the surface of the substrate comprising a thickness in the range of about 500 nanometers to about 50 micrometers.
- Attorney Docket No.: 057862-516001WO 68 The array of any one of paragraphs 54 to 67, wherein the filling material is in a fluid state when applied to the surface of the substrate, and/or wherein the method further comprises solidifying or polymerizing the filling material via heating, UV curing, or cross-linking. 69.
- any one of paragraphs 71 to 75 wherein the feature has a location on the substrate and comprises a plurality of polynucleotide capture probes, wherein a polynucleotide capture probe in the plurality of capture probes comprises a bead barcode and a capture domain, and wherein the capture domain is capable of binding to an analyte.
- a polynucleotide capture probe in the plurality of capture probes comprises a bead barcode and a capture domain, and wherein the capture domain is capable of binding to an analyte.
- every polynucleotide capture probe of the plurality of polynucleotide capture probes on the feature comprises the same bead barcode.
- the array of paragraph 77 wherein for each feature of the plurality of features, the plurality of polynucleotide capture probes on the feature of the plurality of features comprises a different bead barcode than other polynucleotide capture probes on other features of the plurality of features.
- the analyte is mRNA or DNA.
- the capture domain comprises a poly(T) sequence.
- the bead barcode is associated with the specific location of the feature on the substrate. 82.
- kits for the spatial analysis of a biological analyte in a biological sample comprising an array according to any one of paragraphs 35, or 54 to 81, optionally wherein the kit further comprises instructions for use thereof.
- the biological analyte is a nucleic acid.
- the nucleic acid is selected from the group consisting of mRNA, gDNA, rRNA, and tRNA. Attorney Docket No.: 057862-516001WO 85.
- kit comprises a fixative selected from the group consisting of: ethanol, methanol, acetone, formaldehyde, paraformaldehyde-Triton, glutaraldehyde, and combinations thereof.
- kit further comprises one or more staining reagents for staining the biological sample.
- the one or more staining reagents comprises eosin and/or hematoxylin.
- the one or more staining reagents comprises a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.
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Abstract
The present disclosure relates generally to arrays for transcriptomics assays and methods of preparing the same. The disclosure provides arrays having features on a substrate and filling material in the spaces on the substrate that are not occupied by the features. Particularly, the disclosure provides arrays in which the features have a first refractive index at a wavelength and the filling material have a second refractive index at the same wavelength, such that the difference between the first refractive index and the second refractive index is no greater than about 10%. The disclosure also provides methods of preparing such arrays as well as methods of detecting biological analytes using said arrays.
Description
Attorney Docket No.: 057862-516001WO HYBRID MICROBEAD ARRAYS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No.63/380,298, filed October 20, 2022, the disclosure of which is incorporated by reference herein in its entirety, including any drawings. FIELD [0002] The present disclosure relates generally to the field of spatial transcriptomics and particularly to compositions and methods useful for spatially resolving analytes from biological tissues. More particularly, the present disclosure relates to compositions and methods involving hybrid microbead arrays. BACKGROUND [0003] Biological tissues are composed of diverse cells, extracellular matrix, and complex signaling systems organized to interact and execute complex functions. It has been well established that tissue functions essentially rely on the precise spatial organization of cells characterized by distinct molecular profiles. In particular, cells within a tissue of a subject have differences in cell morphology and/or function due to varied levels of biomolecules and metabolites within the different cells. Therefore, determining the spatial distribution of biomolecules can be of great significance for life sciences research, molecular diagnostics and many other applications. In addition to understanding the gene expression profile of a particular cell or tissue, spatial information of biomolecules (e.g., nucleic acids, chemical metabolites, proteins, etc.) within the cell or tissue may also provide valuable information. For example, gene expression profiling of cancer cells can be important for monitoring cancer therapy. [0004] Some spatial transcriptomics platforms involve the transfer and capture of cellular analytes, or proxies thereof, from a tissue section to barcoded capture probes. The capture probes are generally immobilized on a substrate or feature. Features may take a variety of forms, one of which is a bead. Bead microarrays are widely used in biological analyses and multiplexing
Attorney Docket No.: 057862-516001WO applications. [0005] Most bead arrays, however, are unsuitable for imaging with a biological sample (e.g., tissue section) on top, due to the refraction created by the beads. In addition, gaps between the beads cause physical distortions of the biological sample placed on top of the bead array as the sample conforms to the shape and diameter of the beads. Moreover, strong adhesion and retention of the beads to the substrate is difficult to achieve due to limited contact areas between the bead and the substrate. [0006] Therefore, there is a need in the art for arrays that (i) have better adhesion and retention of the features, e.g., beads, on the substrate, (ii) have improved optical transparency, and (iii) provide a suitable surface for biological samples (e.g., tissue samples) placed in contact therewith. SUMMARY [0007] The present disclosure generally relates to, inter alia, compositions and methods for improving spatial transcriptomics platforms. In an aspect, the disclosure relates to methods of preparing an array including: (a) providing a substrate comprising a plurality of features attached to a surface of the substrate, wherein a feature of the plurality of features has a first refractive index at a first wavelength, (b) providing a filling material, wherein the filling material has a second refractive index at the first wavelength, and wherein the difference between the first refractive index and the second refractive index is no greater than about 10%; and (c) applying the filling material to the surface of the substrate such that spaces between the plurality of features attached to the surface of the substrate substantially comprise the filling material. [0008] In some embodiments of the methods of preparing the arrays of the disclosure, the methods further include applying a force to the filling material on the surface of the substrate and/or to the substrate such that the filling material is substantially uniformly distributed across the surface of the substrate. In some embodiments, the force is a centrifugal or mechanical force. [0009] In some embodiments of the methods of preparing the arrays of the disclosure, the methods further include subjecting the filling material on the surface of the substrate to desiccation, wherein an amount of trapped gas in the filling material following desiccation is less than an amount of trapped gas in the filling material prior to desiccation.
Attorney Docket No.: 057862-516001WO [0010] In further embodiments of the methods of preparing the arrays of the disclosure, the substrate includes a plurality of microwells, wherein a microwell of the plurality of microwells includes a feature. In some embodiments, the feature is attached to a surface of the microwell. In some embodiments, the plurality of microwells is etched on the surface of the substrate. In some embodiments, the plurality of microwells is electroplated on the surface of the substrate. In some embodiments, the plurality of microwells is photolithographically deposited on the surface of the substrate. In some embodiments, the surface of the substrate is functionalized with one or more silanes, amines, or a combination thereof. In some embodiments, the substrate is transparent and/or translucent. In some embodiments, the substrate is glass. In some embodiments, the substrate is silica. [0011] In some embodiments of the methods of preparing the arrays of the disclosure, the filling material includes a polymer, optionally wherein the polymer is in the form of a gel, a hydrogel, or a viscous liquid. In some embodiments, the polymer is polydimethylsiloxane (PDMS). [0012] In some embodiments of the methods of preparing the arrays of the disclosure, the filling material is reversibly attached to the substrate. In some embodiments, the filling material forms a layer on the surface of the substrate comprising a thickness in the range of about 500 nanometers to about 50 micrometers. In some embodiments, the filling material is in a fluid state when applied to the surface of the substrate, and the methods further include solidifying or polymerizing the filling material, e.g., via heating, UV curing, or cross-linking. [0013] In some embodiments of the methods of preparing the arrays of the disclosure, the feature is attached to the substrate via heating, gluing, UV curing, or cross-linking. In some embodiments, the feature is attached to the substrate via electrostatic interactions or mechanical fixation. [0014] In some embodiments of the methods of preparing the arrays of the disclosure, the feature is a bead. In some embodiments, the bead has a diameter of about 0.1 µm to about 5 µm, about 1 μm to about 10 μm, about 1 μm to about 20 μm, about 1 μm to about 30 μm, about 1 μm to about 40 μm, about 1 μm to about 50 μm, about 1 μm to about 60 μm, about 1 μm to about 70 μm, about 1 μm to about 80 μm, about 1 μm to about 90 μm, about 90 μm to about 100 μm, about 80 μm to about 100 μm, about 70 μm to about 100 μm, about 60 μm to about 100 μm,
Attorney Docket No.: 057862-516001WO about 50 μm to about 100 μm, about 40 μm to about 100 μm, about 30 μm to about 100 μm, about 20 μm to about 100 μm, or about 10 μm to about 100 μm. [0015] In further embodiments of the methods of preparing the arrays of the disclosure, the feature is composed of a material selected from the group consisting of silica, polystyrene, hydrogel, and a combination thereof. [0016] In some embodiments, the filling material and the composition of the feature are substantially the same, and/or the feature and the filling material have the same refractive index at the first wavelength. In some embodiments, the filling material and the composition of the feature are different. [0017] In some embodiments of the methods of preparing the arrays of the disclosure, the feature has a location on the substrate and the feature includes a plurality of polynucleotide capture probes, such that a polynucleotide capture probe in the plurality of polynucleotide capture probes includes a bead barcode and a capture domain, wherein the capture domain is capable of binding to an analyte. In some embodiments, every polynucleotide capture probe of the plurality of polynucleotide capture probes on the feature includes the same bead barcode. In some embodiments, for each feature of the plurality of features, the plurality of polynucleotide capture probes on the feature includes a different bead barcode than other polynucleotide capture probes on other features of the plurality of features. [0018] In some embodiments, the analyte is mRNA or DNA. [0019] In some embodiments, the capture domain includes a poly(T) sequence. [0020] In some embodiments of the methods of preparing the arrays of the disclosure, the bead barcode of the feature is associated with the location of the feature on the substrate. [0021] In some embodiments of the methods of preparing the arrays of the disclosure, the methods further include determining the sequence of the bead barcode. In some embodiments, the determining includes sequencing. In some embodiments, the sequencing is in situ sequencing. In some embodiments, in situ sequencing is performed via sequencing-by-synthesis (SBS), sequential fluorescence hybridization, sequencing by ligation, nucleic acid hybridization, or high-throughput digital sequencing techniques. [0022] In another aspect, the disclosure provides arrays prepared by the methods of the disclosure.
Attorney Docket No.: 057862-516001WO [0023] In yet another aspect, the disclosure provides methods for detecting a biological analyte in a biological sample including: (a) providing an array having a plurality of features attached to a surface of a substrate prepared by any of the methods of the disclosure, (b) contacting the biological sample with the array, (c) imaging the biological sample to generate an image of the biological sample, (d) incubating the biological sample under conditions wherein the biological analyte binds to a capture probe on a feature of the plurality of features, (e) determining a location of the analyte on the surface of the substrate; and (f) mapping the location of the analyte on the surface of the substrate onto a location in the biological sample using the image of the biological sample. [0024] In some embodiments of the methods for detecting a biological analyte in a biological sample, the determining a location of the analyte on the surface of the substrate includes sequencing. In some embodiments, the sequencing is in situ sequencing. In some embodiments, the in situ sequencing is performed via sequencing-by-synthesis (SBS), sequential fluorescence hybridization, sequencing by ligation, nucleic acid hybridization, or high- throughput digital sequencing techniques. [0025] In some embodiments of the methods for detecting a biological analyte in a biological sample of the disclosure, a distortion in the image of the biological sample resulting from the feature is reduced as compared to a distortion in a reference image of the biological sample, wherein the reference image is generated using a corresponding array that does not include the filling material (e.g., an array having the same features and substrate, but not the filling material) during the imaging. [0026] In a further aspect, the disclosure provides methods for detecting a biological analyte in a biological sample including: (a) contacting the biological sample to an array of the disclosure, (b) imaging the biological sample to generate an image of the biological sample, (c) incubating the biological sample under conditions wherein the biological analyte binds a capture probe on a feature in the plurality of features, (d) determining (i) all or a part of the sequence of the biological analyte specifically bound to the capture probe, or a complement thereof, and (ii) the sequence of the bead barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to identify the location of the analyte on the surface of the substrate; and (e)
Attorney Docket No.: 057862-516001WO mapping the location of the analyte on the surface of the substrate onto a location in the biological sample using the image of the biological sample. [0027] In some embodiments of the methods for detecting a biological analyte in a biological sample of the disclosure, a distortion in the image of the biological sample resulting from the feature is reduced as compared to a distortion in a reference image of the biological sample, wherein the reference image is generated using a corresponding spatial array that does not comprise the filling material during the imaging. [0028] In another aspect, the disclosure provides methods for detecting a biological analyte in a biological sample including: (a) contacting the biological sample to an array prepared by any of the methods of the disclosure, (b) incubating the biological sample under conditions wherein the biological analyte binds a polynucleotide capture probe on a feature in the plurality of features, (c) determining (i) all or a part of the sequence of the biological analyte specifically bound to the polynucleotide capture probe, or a complement thereof, and (ii) the sequence of the bead barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to identify the location of the analyte on the surface of the substrate. [0029] In some embodiments of the methods for detecting a biological analyte of the disclosure, the biological sample includes fresh tissue, frozen tissue, formalin fixed, or formalin- fixed, paraffin-embedded tissue. [0030] In some embodiments of the methods for detecting a biological analyte of the disclosure, the methods further include attaching the biological sample to the array and/or permeabilizing the biological sample to release the biological analyte therefrom, optionally wherein the permeabilizing includes the use of an organic solvent, a detergent, an enzyme, or a combination thereof. In some embodiments, the biological analyte is a nucleic acid. In some embodiments, the nucleic acid is selected from the group consisting of mRNA, gDNA, rRNA, and tRNA. [0031] In some embodiments of the methods for detecting a biological analyte of the disclosure, the methods further include fixing the biological sample. In some embodiments, the fixing the biological sample includes use of a fixative selected from the group consisting of: ethanol, methanol, acetone, formaldehyde, paraformaldehyde-Triton, glutaraldehyde, and combinations thereof.
Attorney Docket No.: 057862-516001WO [0032] In some embodiments of the methods for detecting a biological analyte of the disclosure, the methods further include staining the biological sample, imaging the biological sample, or a combination thereof. In some embodiments, the staining includes the use of eosin and/or hematoxylin. In some embodiments, the staining includes use of a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof. [0033] In some embodiments of the methods for detecting a biological analyte of the disclosure, the feature is a bead. [0034] In a further aspect, the disclosure provides arrays including: (a) a substrate having a plurality of features attached to the substrate, wherein a feature of the plurality of features has a first refractive index at a first wavelength; and (b) a filling material, wherein the filling material has a second refractive index at the first wavelength, and wherein the difference between the first refractive index and the second refractive index is no greater than about 10%, wherein the filling material is disposed on a surface of the substrate where the plurality of features are attached, and wherein the filling material substantially fills the spaces between the plurality of features attached to the surface of the substrate. [0035] In some embodiments of the arrays of the disclosure, the substrate further includes a plurality of microwells, and wherein a microwell of the plurality of microwells comprises a feature of the plurality of features. In some embodiments, the feature is attached to a surface of the microwell. In some embodiments, the plurality of microwells is etched on the surface of the substrate. In some embodiments, the plurality of microwells is electroplated on the surface of the substrate. In some embodiments, the plurality of microwells is photolithographically deposited on the surface of the substrate. In some embodiments, the surface of the substrate is functionalized with one or more silanes, amines, or a combination thereof. In some embodiments, the substrate is transparent and/or translucent. In some embodiments, the substrate is glass. In some embodiments, the substrate is silica. [0036] In some embodiments of the arrays of the disclosure, the filling material comprises a polymer, optionally wherein the polymer is in the form of a gel, a hydrogel, or a viscous liquid. In some embodiments, the polymer is polydimethylsiloxane (PDMS). In some embodiments, the filling material is reversibly attached to the substrate. In some embodiments, the filling material
Attorney Docket No.: 057862-516001WO forms a layer on the surface of the substrate comprising a thickness in the range of about 500 nanometers to about 50 micrometers. In some embodiments of the arrays of the disclosure, the filling material is in a fluid state when applied to the surface of the substrate, and/or the method further comprises solidifying or polymerizing the filling material via heating, UV curing, or cross-linking. [0037] In some embodiments of the arrays of the disclosure, the feature is attached to the substrate via heating, gluing, UV curing, or cross-linking. In some embodiments, the feature is attached to the substrate via electrostatic interactions or mechanical fixation. [0038] In some embodiments of the arrays, the feature is a bead. In some embodiments, the bead has a diameter of about 0.1 µm to about 5 µm, about 1 μm to about 10 μm, about 1 μm to about 20 μm, about 1 μm to about 30 μm, about 1 μm to about 40 μm, about 1 μm to about 50 μm, about 1 μm to about 60 μm, about 1 μm to about 70 μm, about 1 μm to about 80 μm, about 1 μm to about 90 μm, about 90 μm to about 100 μm, about 80 μm to about 100 μm, about 70 μm to about 100 μm, about 60 μm to about 100 μm, about 50 μm to about 100 μm, about 40 μm to about 100 μm, about 30 μm to about 100 μm, about 20 μm to about 100 μm, or about 10 μm to about 100 μm. [0039] In some embodiment of the arrays, the feature is composed of a material selected from the group consisting of silica, polystyrene, hydrogel, and a combination thereof. [0040] In other embodiments of the arrays, the filling material and the composition of the feature are substantially the same, and/or the feature and the filling material have the same refractive index at the first wavelength. In some embodiments, the filling material and the composition of the feature are different. [0041] In some embodiments of the arrays of the disclosure, the feature has a location on the substrate and includes a plurality of polynucleotide capture probes, wherein a polynucleotide capture probe in the plurality of polynucleotide capture probes includes a bead barcode and a capture domain, such that the capture domain is capable of binding to an analyte. In some embodiments, every polynucleotide capture probe of the plurality of polynucleotide capture probes on the feature includes the same bead barcode. [0042] In some embodiments of the arrays, for each feature of the plurality of features, the plurality of polynucleotide capture probes on the feature of the plurality of features includes a
Attorney Docket No.: 057862-516001WO different bead barcode than other polynucleotide capture probes on other features of the plurality of features. [0043] In some embodiments of the arrays, the analyte is mRNA or DNA. In some embodiments of the arrays, the capture domain includes a poly(T) sequence optionally wherein the poly(T) sequence comprises about 20-30 thymidine nucleotides. [0044] In some embodiments of the arrays of the disclosure, the bead barcode is associated with the specific location of the feature on the substrate. [0045] In a further aspect, the disclosure provides kits for spatial analysis of a biological analyte in a biological sample including any of the arrays of the disclosure. In some embodiments of the kits, the kit optionally further includes instructions for use thereof. In some embodiments of the kits of the disclosure, the biological analyte is a nucleic acid. In some embodiments, the nucleic acid is selected from the group consisting of messenger RNA (mRNA), genomic DNA (gDNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). [0046] In some embodiments of the kits, the kits further include one or more fixatives for fixing the biological sample. In some embodiments, the fixative is selected from the group consisting of: ethanol, methanol, acetone, formaldehyde, paraformaldehyde-Triton, glutaraldehyde, and combinations thereof. In some embodiments, the kits further include one or more staining reagents for staining the biological sample. In some embodiments, the staining reagents include eosin and/or hematoxylin. In some embodiments, the one or more staining reagents include a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof. [0047] Each of the aspects and embodiments described herein may be capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect. [0048] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.
Attorney Docket No.: 057862-516001WO BRIEF DESCRIPTION OF THE DRAWINGS [0049] FIG.1 schematically illustrates an exemplary embodiment of the hybrid microbead arrays of the present disclosure. The substrate can be, but is not necessarily, transparent or translucent. [0050] FIG.2 schematically illustrates another exemplary embodiment of the hybrid microbead arrays with microwells of the present disclosure. [0051] FIG.3 schematically shows steps for an exemplary embodiment of a method of preparing a hybrid microbead array of the present disclosure. [0052] FIG.4 shows the optical transparency of a 5µm silica bead array (FIG.4A) and a polydimethylsiloxane (PDMS) filler material deposited on the 5µm silica bead array (FIG.4B). This figure shows that the optical transparency of a 5µm silica bead array (refraction index 1.45) is largely improved after filler material (PDMS) is added to the bead array (refraction index 1.4). DETAILED DESCRIPTION OF THE DISCLOSURE [0053] The present disclosure generally relates to, inter alia, compositions and methods for improving spatial transcriptomics platforms. In particular, the disclosure relates to compositions and methods for improving tissue-imaging quality of bead arrays used in such platforms. More particularly, the present disclosure provides arrays (bead arrays) and methods of preparing arrays that include a filling material. The present disclosure also provides methods for detecting biological analytes, or proxies thereof, using the arrays of the disclosure, as well as kits including said arrays. [0054] Spatial transcriptomic systems may involve the transfer and capture of analytes (e.g., cellular transcripts) from biological samples (e.g. tissue sections) onto bead arrays. The present disclosure provides, inter alia, bead arrays composed of beads embedded in a filling material. The bead arrays of the disclosure include beads and filling material such that the difference between the refractive index of the beads and the refractive index of the filling material is not greater than about 10%. Matching the index of refraction of the beads and the filling material reduces refraction of light passing through the biological sample, such as during imaging for example.
Attorney Docket No.: 057862-516001WO [0055] The bead arrays of the disclosure have several advantages over bead arrays that do not have a filling material. The bead arrays of the disclosure have better adhesion and retention of beads on the substrate, and have improved optical transparency and/or less optical distortion. Moreover, the bead arrays of the disclosure provide an improved surface for the capture of cellular transcripts and other analytes from biological tissues. Furthermore, the filling material enhances the robustness of the bead arrays by providing support for the beads from vibration and other physical interactions, such as etching or scratches on the bead array surface. DEFINITIONS [0056] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. [0057] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, comprising mixtures thereof. “A and/or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”. [0058] The terms “nucleic acid” and “nucleotide” are intended to be consistent with their use in the art and to include naturally-occurring species or functional analogs thereof. Particularly useful functional analogs of nucleic acids are capable of hybridizing to a nucleic acid in a sequence-specific fashion (e.g., capable of hybridizing to two nucleic acids such that ligation can occur between the two hybridized nucleic acids) or are capable of being used as a template for replication of a particular nucleotide sequence. Naturally-occurring nucleic acids generally have a backbone containing phosphodiester bonds. An analog structure can have an alternate backbone linkage including any of a variety of those known in the art. Naturally-occurring nucleic acids generally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid (DNA)) or a ribose sugar (e.g., found in ribonucleic acid (RNA)). A nucleic acid can contain nucleotides
Attorney Docket No.: 057862-516001WO having any of a variety of analogs of these sugar moieties that are known in the art. A nucleic acid can include native or non-native nucleotides. In this regard, a native deoxyribonucleic acid can have one or more bases selected from the group consisting of adenine (A), thymine (T), cytosine (C), or guanine (G), and a ribonucleic acid can have one or more bases selected from the group consisting of uracil (U), adenine (A), cytosine (C), or guanine (G). Useful non-native bases that can be included in a nucleic acid or nucleotide are known in the art. [0059] The term “polynucleotide” refers to a single-stranded multimer of nucleotides which can be from about 2 to about 500 nucleotides in length. Polynucleotides can be synthetic, made enzymatically (e.g., via polymerization), or using a “split-pool” method. Polynucleotides can include ribonucleotide monomers (i.e., can be oligoribonucleotides) and/or deoxyribonucleotide monomers (i.e., oligodeoxyribonucleotides). In some examples, polynucleotides can include a combination of both deoxyribonucleotide monomers and ribonucleotide monomers in the polynucleotide (e.g., random or ordered combination of deoxyribonucleotide monomers and ribonucleotide monomers). A polynucleotide can be 4 to 10, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 500 nucleotides or more in length, for example. Polynucleotides can include one or more functional moieties that are attached (e.g., covalently or non-covalently) to the multimer structure. For example, a polynucleotide can include one or more detectable labels (e.g., a radioisotope or fluorophore). [0060] A “capture probe” when used in reference to a nucleic acid or sequence of a nucleic acids, is intended as a semantic identifier for the nucleic acid or sequence in the context of a method or composition, and does not limit the structure or function of the nucleic acid or sequence beyond what is expressly indicated. [0061] The term “barcode” is used herein to refer to a label, or identifier, that conveys or is capable of conveying information (e.g., information about an analyte in a sample, a bead, and/or a nucleic acid barcode molecule). A barcode can be part of an analyte or nucleic acid barcode molecule, or independent of an analyte or nucleic acid barcode molecule. A barcode can be attached to an analyte or nucleic acid barcode molecule in a reversible or irreversible manner. A particular barcode can be unique relative to other barcodes. Barcodes can have a variety of different formats. For example, barcodes may include polynucleotide barcodes, random nucleic
Attorney Docket No.: 057862-516001WO acid and/or amino acid sequences, and synthetic nucleic acid and/or amino acid sequences. A barcode can be attached to an analyte or to another moiety or structure in a reversible or irreversible manner. A barcode can be added to, for example, a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before or during sequencing of the sample. Barcodes can allow for or facilitate identification and/or quantification of individual sequencing- reads. In some embodiments, a barcode can be configured for use as a fluorescent barcode. For example, in some embodiments, a barcode can be configured for hybridization to fluorescently labeled oligonucleotide capture probes. Barcodes may be configured to spatially resolve molecular components found in biological samples, for example, at single-cell resolution (e.g., a barcode can be or can include a “spatial barcode”). In some embodiments, a barcode includes two or more sub-barcodes that together function as a single barcode. For example, a polynucleotide barcode may include two or more polynucleotide sequences (e.g., sub-barcodes). In some embodiments, the two or more sub-barcodes are separated by one or more non-barcode sequences. In some embodiments, the two or more sub-barcodes are not separated by non- barcode sequences. [0062] The term “cell” refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell, cell culture, or cell line, without regard to the number of transfers or passages in culture. It should be understood that not all progeny are exactly identical to the parental cell. This is because certain modifications may occur in succeeding generations due to either mutation (e.g., deliberate or inadvertent mutations) or environmental influences (e.g., methylation or other epigenetic modifications), such that progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein, so long as the progeny retain the same functionality as that of the originally cell, cell culture, or cell line. [0063] As used herein, the term “attach,” and grammatical derivatives thereof, refer to affixing, securing, associating, fastening, adhering, bonding, or connecting by a bond, link, force (e.g., magnetic force), or tie in order to keep two or more elements together. The term “attach” encompasses either direct or indirect attachments, and also encompasses both covalent attachments and noncovalent attachments. When used with respect to joining a feature to a substrate, the term includes but is not limited to fixing, securing, associating, fastening, or
Attorney Docket No.: 057862-516001WO connecting the feature directly or indirectly to the substrate. [0064] The term “susbtantially” is generally used to indicate approximation and refers to “largely but not necessarily wholly that which is specified.” In the present disclosure, the term“substantially fills,” when used with respect to filling spaces between the features attached to the surface of the substrate, generally refers to applying a volume of the filling material into the spaces between the features such that the volume fills (e.g., occupies) substantially all of the spaces, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or any value in between, of the spaces between the features attached to the surface. In some embodiments, the filling material occupies from 80% to 99%, from 85% to 98%, from 90% to 97%, or from 95% to 96% of the spaces between the features attached to the surface of the substrate. In addition, when the term “substantially fills” is used to refer to a particular space (i.e. one individual space), it generally refers to applying a volume of the filling material into substantially all the particular space, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or any value in between, of the one individual space. In some embodiments, the filling material occupies from 80% to 99%, from 85% to 98%, from 90% to 97%, or from 95% to 96% of the one individual space. [0065] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. In some embodiments, the term “about” indicates the designated value ± up to 10%, ± up to 5%, ± up to 2%, or ± up to 1%. [0066] It is understood that aspects and embodiments of the disclosure described herein include "comprising", "consisting", and "consisting essentially of" aspects and embodiments. As used herein, "comprising" is synonymous with "including", "containing", or "characterized by", and is inclusive or open-ended and does not exclude additional, unrecited elements or method
Attorney Docket No.: 057862-516001WO steps. As used herein, "consisting of" excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term "comprising", particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or steps. [0067] Headings, e.g., (a), (b), (i) etc., are presented merely for ease of reading the specification and claims. The use of headings in the specification or claims does not require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented. [0068] Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order. [0069] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
Attorney Docket No.: 057862-516001WO COMPOSITIONS OF THE DISCLOSURE Arrays and Filling Material [0070] The present disclosure provides, inter alia, arrays for use in spatial transcriptomic analysis. The arrays of the disclosure can include (a) a substrate having a plurality of features attached to the substrate, and (b) a filling material such that the filling material substantially fills the spaces between the plurality of features. [0071] In some embodiments, the features have a first refractive index at a first wavelength (e.g., 1.45) and the filling material has a second refractive index (e.g., 1.4) at the first wavelength. In some embodiments, the first refractive index and the second refractive index are the same. [0072] As used herein, the refractive index can be a dimensionless value representing the relative ratio of the speed of light in a medium (e.g., a filling material) relative to the speed of light in a vacuum. Refractive index can be used to describe the phase velocity of light through a medium, e.g., how much the path of light is refracted when entering a medium (e.g., a filling material). The refractive index of a sample is dependent on temperature and wavelength. Refractive index can be measured using known methods in the art such as, for example, with an Abbe refractometer, an ellipsometer, or a prism coupler refractometer. [0073] In some embodiments, the first refractive index and the second refractive index are different at the same (e.g., a first) wavelength. In some embodiments, the first wavelength is a wavelength of visible light. In some exemplary embodiments, the first wavelength is in the range of about 380 to about 700 nm, or a subrange therein. In some embodiments, the difference between the first refractive index and the second refractive index is no greater than about 10%. In some embodiments, the difference is no greater than about 1%, no greater than 1.5%, no greater than about 2%, no greater than about 2.5%, no greater than about 3%, no greater than about 3.5%, no greater than about 4%, no greater than about 4.5%, no greater than about 5%, no greater than about 5.5%, no greater than about 6%, no greater than about 6.5%, no greater than about 7%, no greater than about 7.5%, no greater than about 8%, no greater than about 8.5%, no greater than about 9%, no greater than about 9.5%, no greater than about 10% or any values in between. [0074] In some embodiments, the features have a first refractive index of about 1.4, about
Attorney Docket No.: 057862-516001WO 1.41, about 1.42, about 1.43, about 1.44, about 1.45, about 1.46, about 1.47, about 1.48, or about 1.49 or within any range having any two of these values as endpoints. In some embodiments, the filling material has a second refractive index about 1.4, about 1.41, about 1.42, about 1.43, about 1.44, about 1.45, about 1.46, about 1.47, about 1.48, or about 1.49 or within any range having any two of these values as endpoints. [0075] The filling material of the arrays of the disclosure can be made from various types of material. The filling material can be made of one or more polymers. The polymer can be in the form of gels, hydrogels, or viscous liquids. In some embodiments, the polymer is polydimethylsiloxane (PDMS), polyethylene, polypropylene, PET, PMMA, or a combination thereof. [0076] The filling material of the arrays of the disclosure can have a thickness in the range of about 500 nanometers to about 50 micrometers when disposed on the substrate. In some embodiments, the thickness of the filling material on the surface of the substrate is in the range of about 400 nanometers to about 45 micrometers. In some embodiments, the thickness of the filling material on the surface of the substrate is in the range of about 300 nanometers to about 40 micrometers, or in the range of about 200 nanometers to about 35 micrometers, or in the range of about 100 nanometers to about 30 micrometers. In some embodiments, the thickness of the filling material on the surface of the substrate can correspond to the size, volume, circumference and/or diameter of the beads of the array. In one embodiment, the thickness of the filling material can correspond to an amount of filling material needed to substantially cover or envelope the beads of the array. [0077] The filling material of the disclosure can be in a fluid state when applied to the surface of the substrate. In some embodiments, the filling material is solidified after application to the substrate. In some embodiments, the filling material is polymerized after application to the substrate. In some embodiments, the filling material is gelled after application to the substrate. The filling material can be gelled, solidified or polymerized by any suitable method known in the art. In some embodiments, the filling material is solidified or polymerized by heating. In some embodiments, the filling material is solidified or polymerized by UV curing. In some embodiments, the filling material is solidified or polymerized by cross-linking. In some embodiments, the filling material is solidified or polymerized by adding an initiator, adding
Attorney Docket No.: 057862-516001WO shear, or changing the pH. [0078] In some embodiments, the filling material covers or substantially covers all spaces in between the features. In some embodiments, the filling material covers the top of the features on the array as shown, for example, in FIG.1 or FIG 2. In some embodiments, the filling material partially covers the features of the array. [0079] The filling material can be permanently attached to the substrate or can be reversibly attached to the substrate. In some embodiments, the filling material is removed when needed. In some embodiments, the filling material is dissolved and removed from the array after certain processes. [0080] In some embodiments, the filling material is subjected to desiccation in order to remove or reduce the formation of air bubbles during the filling process. In some embodiments, the filling material is subjected to a negative pressure (e.g., vacuum) to remove or reduce formation of air bubbles during the filling process. [0081] In an array, a specific arrangement of a plurality of features can be either irregular or can form a regular pattern. Individual features on the array can differ from one another based on their relative spatial locations. In some embodiments, a plurality of the features of the disclosure are spatially ordered in an array and each feature of the plurality of the features is associated with a unique spatial location on the array. [0082] In some embodiments, the arrays of the disclosure are designed to accommodate a large number of features. For example, the array can include at least 5,000 features, for example at least 10,000 features, at least 20,000 features, at least 50,000 features, at least 100,000 features, at least 500,000 features, at least 1,000,000 features, at least 2,000,000 features, at least 5,000,000 features, or at least 10,000,000 features, or more. [0083] In some embodiments, the plurality of features includes a single type of feature (e.g., substantially uniform in volume, shape, and other physical properties, such as translucence). In some embodiments, the plurality of features includes two or more types of different features. [0084] The arrays of the disclosure can include arrays prepared by any of the methods of the disclosure discussed below.
Attorney Docket No.: 057862-516001WO Features [0085] The arrays described herein include features which are designed and/or configured to act as supports or repositories for various molecular entities or proxies thereof used in tissue sample analysis. [0086] The features may be formulated into various shapes and dimensions depending on the context of intended use. Examples of features include, but are not limited to, a bead, a spot of any two- or three-dimensional geometry (e.g., an ink jet spot, a masked spot, a square on a grid), a microwell, and a hydrogel pad. In some preferred embodiments, the features are beads. In some preferred embodiments, the features are microbeads (i.e., beads having a largest dimension of less than one thousand micrometers). [0087] In some embodiments, the feature is a microbead. The microbead may have an average diameter of about 0.1 micrometer (micron) to about 50 micrometers. In some embodiments, the microbead may have an average diameter of about 0.1 micrometer (micron) to about 100 micrometers. In some embodiments, the microbead may have an average diameter of about 0.1 micrometer, about 0.2 micrometers, about 0.3 micrometers, about 0.4 micrometers, about 0.5 micrometers, about 0.6 micrometers, about 0.7 micrometers, about 0.8 micrometers, or about 0.9 micrometers. In some embodiments, the microbead may have an average diameter of about 1 micron, about 2 micrometers, about 3 micrometers, about 4 micrometers, about 5 micrometers, about 6 micrometers, about 7 micrometers, about 8 micrometers, about 9 micrometers, about 10 micrometers, about 11 micrometers, about 12 micrometers, about 13 micrometers, about 14 micrometers, about 15 micrometers, about 16 micrometers, about 17 micrometers, about 18 micrometers, about 19 micrometers, about 20 micrometers, about 25 micrometers, about 30 micrometers, about 35 micrometers, about 40 micrometers, about 45 micrometers, about 50 micrometers, about 60 micrometers, about 70 micrometers, about 80 micrometers, about 90 micrometers, about 100 micrometers or any other values in between. [0088] In some embodiments, the bead (e.g., microbead) may have a diameter of about 0.1 micrometer to about 5 micrometers, about 1 micrometer to about 10 micrometers, about 1 micrometer to about 20 micrometers, about 1 micrometer to about 30 micrometers, about 1 micrometer to about 40 micrometers, about 1 micrometer to about 50 micrometers, about 1 micrometer to about 60 micrometers, about 1 micrometer to about 70 micrometers about 1
Attorney Docket No.: 057862-516001WO micrometer to about 80 micrometers about 1 micrometer to about 90 micrometers about 1 micrometer to about 100 micrometers or any other ranges in between. [0089] In some preferred embodiments, the bead (e.g., microbead) has a diameter of about 0.1 µm, about 0.5 µm, about 1 µm, about 2µm, about 3µm, about 4 µm, about 5 µm, about 6 µm, about 7 µm, about 8 µm, about 9 µm, about 10 µm, about 11 µm, or about 12 µm. [0090] The features of the disclosure can be in the form of a polymer. In some embodiments, the polymer can be in the form of a hydrogel, a gel, a viscous liquid or other forms. [0091] The features of the disclosure can be composed of various materials. Non-limiting examples of materials include silica, polystyrene, carboxyl-modified polystyrene, polystyrene/2% divinylbenzene, polystyrene/10% divinylbenzene, polystyrene/55% divinylbenzene, polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), or any combination thereof. In some embodiments, the features are composed of silica, polystyrene, hydrogel or a combination thereof. In some embodiments, the feature may be further modified by silane deposition and/or polymer adsorption. [0092] In some embodiments, the features (e.g., beads) are optically transparent or translucent. In some embodiments, the features (e.g., beads) are optically transparent or translucent when the substrate is imaged at a first wavelength. [0093] The features of the arrays of the disclosure can be attached to a substrate via a variety of methods. Non-limiting examples of how features can be attached to the substrate include chemical bonding (e.g., via a covalent bond), electrostatic interactions, mechanical fixation, or other methods. In some embodiments, the features can be attached to the substrate via heating, gluing, UV curing, cross-linking or by functionalization e.g., via salines or amines, or by other methods known to those skilled in the art. [0094] In some embodiments, the features of the disclosure can be amine modified and arrayed with EDC-coupling on N-hydroxysuccinimide glass slides. [0095] In some embodiments, the features are attached to the top surface of the substrate. In some embodiments, the features are located partially or completely within microwells in the substrate. In some embodiments, each microwell in the substrate includes a single feature. [0096] In some embodiments, some or all the features of the disclosure are functionalized
Attorney Docket No.: 057862-516001WO for analyte capture. For example, the features may include capture probes (including e.g., capture domains) for analyte capture. A skilled artisan in the art will understand that the term capture probe generally refers to any molecule capable of capturing (directly or indirectly) and/or labelling an analyte of interest in or from a biological sample. In some embodiments, the capture probes may include a nucleic acid binding moiety (NABM), which can be small molecules, aptamers, proteins, or oligonucleotides. In some embodiments, the capture probes are polynucleotide capture probes. In some embodiments, the capture probes may include ribonucleotides and/or deoxyribonucleotides as well as synthetic nucleotide residues that are capable of participating in Watson-Crick type or analogous base pair interactions. In some embodiments, the capture probes may include a conjugate (e.g., an oligonucleotide-antibody conjugate). [0097] In some embodiments, a feature can occupy a discrete location on the substrate and include a plurality of capture probes (e.g., polynucleotide capture probes) such that the capture probes on the feature include a bead barcode and a capture domain capable of binding or hybridizing to an analyte or a proxy thereof. [0098] The features may include a plurality of capture probes at an average density of about 500 to about 100,000 capture probes per micrometer2. In some embodiments, the average density is about 1,000 to about 100,000 capture probes per micrometer2. In some embodiments, the average density is about 10,000 to about 80,000 capture probes per micrometer2. In some embodiments, the average density is about 25,000 to about 60,000 capture probes per micrometer2. [0099] In some embodiments, the features may include a plurality of capture probes at an average density of at least about 500 molecules per μm2, at least about 1000 molecules per μm2, at least about 1500 molecules per μm2, at least about 2000 molecules per μm2, at least about 5000 molecules per μm2, at least about 10,000 molecules per μm2, at least about 15,000 molecules per μm2, at least about 20,000 molecules per μm2, at least about 25,000 molecules per μm2, at least about 30,000 molecules per μm2, at least about 35,000 molecules per μm2, at least about 40,000 molecules per μm2, at least about 45,000 molecules per μm2, at least about 50,000 molecules per μm2, at least about 55,000 molecules per μm2, at least about 60,000 molecules per μm2, at least about 65,000 molecules per μm2, at least about 70,000 molecules per μm2, at least
Attorney Docket No.: 057862-516001WO about 75,000 molecules per μm2, at least about 80,000 molecules per μm2, at least about 85,000 molecules per μm2, at least about 90,000 molecules per μm2, at least about 95,000 molecules per μm2, at least about 100,000 molecules per μm2, or any value in between. Capture probes [0100] The features of the disclosure may include a plurality of capture probes. The capture probes of the disclosure may be directly or indirectly attached, incorporated, embedded, and/or affixed to the features. In some embodiments, the feature is a hydrogel having a plurality of moieties and the capture probe is attached to a moiety of the plurality of moieties within the hydrogel. [0101] In some embodiments, the capture probe may include a barcode sequence (e.g., a spatial barcode, a bead barcode) and/or a capture domain. The bead barcode can be specific to a single bead so that different beads have different bead barcodes. In some embodiments, all capture probes on a bead have the same bead barcode. In some embodiments, all beads on a bead array have different bead barcodes. [0102] The capture probes of the disclosure may include capture domains. The capture domain may be capable of binding (e.g., hybridizing to) an analyte or a proxy thereof. The capture domain may include a nucleic acid sequence (e.g., a poly(T) sequence) capable of binding to a poly(A) tail of an mRNA and/or to a poly(A) homopolymeric sequence present in genomic DNA. In some embodiments, a homopolymeric sequence is added to an mRNA molecule or a genomic DNA molecule using a terminal transferase enzyme in order to produce a molecule that has a poly(A) or poly(T) sequence. For example, a poly(A) sequence can be added to an analyte (e.g., a fragment of genomic DNA) thereby making the analyte capable of capture by a poly(T) capture domain. In some embodiments, a capture domain includes a poly(T) sequence, a random sequence, a gene specific sequence, a degenerate sequence, or a combination thereof. In some embodiments, the random sequence includes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more random nucleotides. In some embodiments, the random sequence includes a random hexamer. In some embodiments, the random sequence includes a random decamer. [0103] In some embodiments, the capture probe may include a unique molecular identifier (UMI). UMIs can be used for determining the number of unique molecules in a nucleic acid
Attorney Docket No.: 057862-516001WO library (e.g., a NGS library) as described below in more detail. [0104] Each capture probe can optionally include at least one cleavage domain. The cleavage domain represents a portion of the capture probe that is used to reversibly attach the capture probe to a feature. Further, one or more segments or regions of the capture probe can optionally be released from the feature by cleavage of the cleavage domain. As an example, spatial (e.g., bead) barcodes and/or universal molecular identifiers (UMIs) can be released by cleavage of the cleavage domain. In some embodiments, the capture probe may be optionally coupled to a feature by a cleavage domain, such as a disulfide linker. [0105] In some embodiments, a cleavage domain is absent from the capture probe. Examples of substrates with attached capture probes lacking a cleavage domain are described for example in Macosko et al., (2015) Cell 161, 1202–1214. [0106] In some embodiments, each capture probe of the disclosure may optionally include at least one, at least two, a least three, or at least four functional domains or regions. Each functional domain or region generally includes a functional nucleotide sequence for a downstream analytical step in the overall analysis procedure. For example, a capture probe of the disclosure can include (i) a sequencing domain, (ii) a PCR adaptor, (iii) a primer binding site and/or (iv) a capture region capable of binding to a target analyte or a proxy thereof. [0107] In some embodiments, the capture probe can include adapters that are useful for subsequent downstream processing. Examples of such as an adapter include a sequencer specific flow cell attachment sequence, e.g., a P5 sequence and/or a P7 sequence, as well as sequencing primer sequences, e.g., a R1 primer binding site and/or a R2 primer binding site. Spatial barcodes [0108] In general, the spatial barcode (e.g., bead barcodes) of the disclosure includes a contiguous nucleic acid segment or at least two non-contiguous nucleic acid segments that function as a label or identifier that conveys or is capable of conveying spatial information. In some embodiments, a capture probe can include a spatial barcode (e.g., spatial bead barcode) that possesses a spatial aspect, where the bead barcode is associated with a particular location within an array or a particular location on a substrate. Thus, a bead barcode can be a spatial barcode once the bead is positioned on the substrate and associated with a unique position thereon. A spatial barcode can be incorporated within the capture probe for use in barcoding a target analyte
Attorney Docket No.: 057862-516001WO or a proxy thereof. In some embodiments, the functional sequences of a barcode can generally be designed or selected for compatibility with any of a variety of different sequencing systems, e.g., 454 Sequencing, Ion Torrent Proton or PGM, Illumina sequencing instruments, PacBio, Nanopore, etc., and the requirements thereof. In some embodiments, functional sequences can be designed or selected for compatibility with non-commercialized sequencing systems. [0109] A spatial barcode may be part of an analyte, or independent from an analyte (e.g., part of the capture probe). A spatial barcode can be a tag attached to an analyte (e.g., a nucleic acid molecule) or a combination of a tag in addition to an endogenous characteristic of the analyte (e.g., size of the analyte). [0110] In some embodiments, the spatial barcode can be common to all of the capture probes attached to a given feature (feature barcode). In some embodiments, a spatial barcode can be unique. In some embodiments where the spatial barcode is unique, the spatial barcode functions both as a spatial barcode and as a unique molecular identifier (UMI), associated with one particular capture probe. In some embodiments, the spatial barcode and UMI are separate nucleic acid sequences and their functions within a capture probe do not overlap. [0111] Spatial barcodes can have a variety of different formats. For example, spatial barcodes can include polynucleotide spatial barcodes, random nucleic acid and/or amino acid sequences, and synthetic nucleic acid and/or amino acid sequences. In some embodiments, a spatial barcode is attached to an analyte in a reversible or irreversible manner. In some embodiments, a spatial barcode is added to, for example, a fragment of a DNA or RNA sample before, during, and/or after downstream analyses (e.g., sequencing) of the sample. In some embodiments, a spatial barcode is designed or selected to allow for identification and/or quantification of individual sequencing-reads. In some embodiments, a spatial barcode is used as a fluorescent barcode for which fluorescently labeled oligonucleotide capture probes hybridize and/or attach to the spatial barcode. [0112] In some embodiments, the spatial barcode is a nucleic acid sequence that does not substantially hybridize to analyte nucleic acid molecules in a biological sample. In some embodiments, the spatial barcode has less than 80% sequence identity (e.g., less than 70%, 60%, 50%, or less than 40% sequence identity) to the nucleic acid sequences across a substantial part (e.g., 80% or more) of the nucleic acid molecules in the biological sample.
Attorney Docket No.: 057862-516001WO [0113] In instances where multiple capture probes are attached to a feature, one or more spatial barcode sequences of the multiple capture probes can include sequences that are the same for all capture probes coupled to the feature, and/or sequences that are different across all capture probes coupled to the feature. [0114] In some embodiments, capture probes attached to a single feature can include identical (or common) spatial barcode sequences, different spatial barcode sequences, or a combination of both. Capture probes attached to a feature can include multiple sets of capture probes. Capture probes of a given set can include identical spatial barcode sequences. The identical spatial barcode sequences can be different from spatial barcode sequences of capture probes of another set. In some embodiments, the identical spatial barcode sequences can be different from spatial barcode sequences of capture probes attached to another feature. [0115] The plurality of capture probes can include spatial barcode sequences (e.g., nucleic acid barcode sequences) that are associated with specific locations on a spatial array. For example, a first plurality of capture probes can be associated with a first region, based on a spatial barcode sequence common to the capture probes within the first region, and a second plurality of capture probes can be associated with a second region, based on a spatial barcode sequence common to the capture probes within the second region. The second region may or may not be associated with the first region. Additional pluralities of capture probes can be associated with spatial barcode sequences common to the capture probes within other regions. In some embodiments, the spatial barcode sequences can be the same across a plurality of capture probe molecules. [0116] In some embodiments, multiple different spatial barcodes are incorporated into a single feature. For example, a mixed but known set of spatial barcode sequences can provide a stronger address or attribution of the spatial barcodes to a given spot or location, by providing duplicate or independent confirmation of the identity of the location. In some embodiments, the multiple spatial barcodes represent increasing specificity of the location of the particular feature on the array. Capture Domains [0117] A capture probe of the disclosure can include at least one capture domain. The “capture domain” can be an oligonucleotide, a polypeptide, a small molecule, or any
Attorney Docket No.: 057862-516001WO combination thereof, that binds specifically (e.g., hybridizes) to a desired analyte or class of analytes, or a proxy thereof. In some embodiments, a capture domain can be used to directly or indirectly capture or detect a desired analyte. [0118] In some embodiments, the capture domain is a functional nucleic acid sequence configured to interact with one or more analytes or proxies thereof, such as one or more different types of nucleic acids (e.g., RNA molecules and DNA molecules). In some embodiments, the functional nucleic acid sequence can include an N-mer sequence (e.g., a random N-mer sequence), which N-mer sequences are configured to interact with a plurality of DNA molecules. In some embodiments, the functional sequence can include a poly(T) sequence. In some embodiments, poly(T) sequences are configured to interact with messenger RNA (mRNA) molecules via the poly(A) tail of an mRNA transcript. In some embodiments, poly(T) sequences are configured to interact with a target nucleic acid (e.g., RTL probe) via a poly(A) sequence contained within the target nucleic acid. In some embodiments, the functional nucleic acid sequence is the binding target of a protein (e.g., a transcription factor, a DNA binding protein, or a RNA binding protein), where the analyte of interest is a protein. [0119] In some embodiments, the capture domain is capable of priming a reverse transcription reaction to generate cDNA that is complementary to captured RNA molecules. In some embodiments, the capture domain of the capture probe can prime a DNA extension (polymerase) reaction to generate DNA that is complementary to the captured DNA molecules. In some embodiments, the capture domain can template a ligation reaction between captured DNA molecules and a surface capture probe that is directly or indirectly immobilized on the substrate. In some embodiments, the capture domain can be ligated to one strand of the captured DNA molecules. For example, SplintR ligase along with RNA or DNA sequences (e.g., degenerate RNA) can be used to ligate a single-stranded DNA or RNA to the capture domain. In some embodiments, ligases with RNA-templated ligase activity, e.g., SplintR ligase, T4 RNA ligase 2 or KOD ligase, can be used to ligate a single-stranded DNA or RNA to the capture domain. In some embodiments, a capture domain includes a splint oligonucleotide sequence. In some embodiments, a capture domain captures a splint oligonucleotide. [0120] In some embodiments, the capture domain is located at the 3’ end of the capture probe and includes a free 3’ end that can be extended, e.g., by template dependent
Attorney Docket No.: 057862-516001WO polymerization, to form an extended capture probe as described herein. In some embodiments, the capture domain includes a nucleotide sequence that is capable of hybridizing to nucleic acid, e.g., RNA or other analyte, present in the cells of the biological sample contacted with the array. In some embodiments, the capture domain can be selected or designed to bind selectively or specifically to a target nucleic acid. For example, the capture domain can be selected or designed to capture mRNA by way of hybridization to the mRNA poly(A) tail. Thus, in some embodiments, the capture domain includes a poly(T) DNA oligonucleotide, e.g., a series of consecutive deoxythymidine residues linked by phosphodiester bonds, which is capable of hybridizing to the poly(A) tail of mRNA. In some embodiments, the capture domain can include nucleotides that are functionally or structurally analogous to a poly(T) tail. For example, a poly(U) oligonucleotide or an oligonucleotide included of deoxythymidine analogues. In some embodiments, the capture domain includes at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides (e.g., T or U). In some embodiments, the capture domain includes at least 25, 30, or 35 nucleotides (e.g., T or U). [0121] In some embodiments, random sequences, e.g., random hexamers, decamers, or similar sequences, can be used to form all or a part of the capture domain. For example, random sequences can be used in conjunction with poly(T) (or poly(T) analogue) sequences. Thus, where a capture domain includes a poly(T) (or a “poly(T)-like”) oligonucleotide, it can also include a random oligonucleotide sequence (e.g., “poly(T)-random sequence” capture probe). This can, for example, be located 5’ or 3’ of the poly(T) sequence, e.g., at the 3’ end of the capture domain. The poly(T)-random sequence capture probe can facilitate capture of the mRNA poly(A) tail. In some embodiments, the capture domain can be an entirely random sequence. In some embodiments, degenerate capture domains can be used. [0122] The capture domain can be based on a particular gene sequence or particular motif sequence or common/conserved sequence, that it is designed to capture (i.e., a sequence-specific capture domain). Thus, in some embodiments, the capture domain can bind (e.g., is configured for binding) selectively to a desired sub-type or subset of nucleic acid, for example a particular type of RNA, such as mRNA, rRNA, tRNA, SRP RNA, tmRNA, snRNA, snoRNA, SmY RNA, scaRNA, gRNA, RNase P, Rnase MRP, TERC, SL RNA, aRNA, cis-NAT, crRNA, lncRNA, miRNA, piRNA, siRNA, shRNA, tasiRNA, rasiRNA, 7SK, eRNA, ncRNA or other types of
Attorney Docket No.: 057862-516001WO RNA. In a non-limiting example, the capture domain can be capable of, or configured for, binding selectively to a desired subset of ribonucleic acids, for example, microbiome RNA, such as 16S rRNA. [0123] In some embodiments, a capture domain includes an “anchor” or “anchoring sequence”, which is a sequence of nucleotides that is designed to ensure that the capture domain hybridizes to the intended analyte, or proxy thereof. In some embodiments, an anchor sequence includes a sequence of nucleotides, including a 1-mer, 2-mer, 3-mer or longer sequence. In some embodiments, the sequence is random. For example, a capture domain including a poly(T) sequence can be designed to capture an mRNA. In such embodiments, an anchoring sequence can include a random 3-mer (e.g., GGG) that helps ensure that the poly(T) capture domain hybridizes to an mRNA. In some embodiments, an anchoring sequence can be VN, N, or NN. Alternatively, the sequence can be designed using a specific sequence of nucleotides. In some embodiments, the anchor sequence is at the 3’ end of the capture domain. In some embodiments, the anchor sequence is at the 5’ end of the capture domain. [0124] In some embodiments, capture domains of capture probes are blocked prior to contacting the biological sample with the array, and blocking probes may be used when the nucleic acids in the biological sample are modified prior to capture on the array. In some embodiments, the blocking probe is used to block or modify the free 3’ end of the capture domain. In some embodiments, blocking probes can be hybridized to the capture probes to mask the free 3’ end of the capture domain, e.g., hairpin probes, partially double stranded probes, or complementary sequences. In some embodiments, the free 3’ end of the capture domain can be blocked by chemical modification, e.g., addition of an azidomethyl group as a chemically reversible capping moiety such that the capture probes do not include a free 3’ end. Blocking or modifying the capture probes, particularly at the free 3’ end of the capture domain, prior to contacting the biological sample with the array, prevents modification of the capture probes, e.g., prevents the addition of a poly(A) tail to the free 3’ end of the capture probes. [0125] Non-limiting examples of 3’ modifications include dideoxy C-3’ (3’-ddC), 3’ inverted dT, 3’ C3 spacer, 3’Amino, and 3’ phosphorylation. In some embodiments, the nucleic acid in the biological sample can be modified such that it can be captured by the capture domain. For example, an adaptor sequence (including a binding domain capable of binding to the capture
Attorney Docket No.: 057862-516001WO domain of the capture probe) can be added to the end of the target nucleic acid, e.g., fragmented genomic DNA. In some embodiments, this is achieved by ligation of the adaptor sequence or extension of the nucleic acid. In some embodiments, an enzyme is used to incorporate additional nucleotides at the end of the nucleic acid sequence, e.g., a poly(A) tail. In some embodiments, the capture probes can be reversibly masked or modified such that the capture domain of the capture probe does not include a free 3’ end. In some embodiments, the 3’ end is removed, modified, or made inaccessible so that the capture domain is not susceptible to the process used to modify the nucleic acid of the biological sample, e.g., ligation or extension. [0126] In some embodiments, the capture domain of the capture probe is modified to allow the removal of any modifications of the capture probe that occur during modification of the nucleic acid molecules of the biological sample. In some embodiments, the capture probes can include an additional sequence downstream of the capture domain, e.g., 3’ to the capture domain, namely a blocking domain. [0127] In some embodiments, the capture domain of the capture probe can be a non- nucleic acid domain. Examples of suitable capture domains that are not exclusively nucleic-acid based include, but are not limited to, proteins, peptides, aptamers, antigens, antibodies, and molecular analogs that mimic the functionality of any of the capture domains described herein. Unique molecular Identifiers (UMI) [0128] The capture probes of the disclosure can include one or more (e.g., two or more, three or more, four or more, five or more) Unique Molecular Identifiers (UMIs). A unique molecular identifier is a contiguous nucleic acid segment or two or more non-contiguous nucleic acid segments that function as a label or identifier for a particular analyte, or for a capture probe that binds a particular analyte or a proxy thereof (e.g., via the capture domain). [0129] A UMI can include one or more specific polynucleotides sequences, one or more random nucleic acid and/or amino acid sequences, and/or one or more synthetic nucleic acid and/or amino acid sequences. [0130] In some embodiments, the UMI is a nucleic acid sequence that does not substantially hybridize to analyte nucleic acid molecules in a biological sample. In some embodiments, the UMI has less than 80% sequence identity (e.g., less than 70%, 60%, 50%, or less than 40% sequence identity) to the nucleic acid sequences across a substantial part (e.g.,
Attorney Docket No.: 057862-516001WO 80% or more) of the nucleic acid molecules in the biological sample. These nucleotides can be completely contiguous, i.e., in a single stretch of adjacent nucleotides, or they can be separated into two or more separate subsequences that are separated by one or more nucleotides. [0131] In some embodiments, a UMI is attached to an analyte in a reversible or irreversible manner. In some embodiments, a UMI is added to, for example, a fragment of a DNA or RNA sample before or during, sequencing of the analyte. In some embodiments, a UMI allows for identification and/or quantification of individual sequencing-reads. In some embodiments, a UMI is a used as a fluorescent barcode for which fluorescently labeled oligonucleotide probes hybridize to the UMI. Functionally, UMIs allow for demultiplexing of sequencing reads and biases that may be introduced through downstream processing (e.g., PCR amplification). For example, each biomolecule captured by a capture probe on a feature of a spatial bead array should contain a distinct UMI that is distinguishable from other UMIs on the spatial bead array. Typically, if two sequencing reads contain the same UMI, they are discarded from the data set. Substrates [0132] Provided in the present disclosure are substrates upon which features are immobilized. A substrate may be designed and/or configured to function as a support for direct or indirect attachment of the features of the disclosure. [0133] Features may be directly or indirectly attached, embedded, or affixed to a substrate. In some embodiments, the features are not directly or indirectly attached embedded, or affixed to a substrate, but instead, for example, are disposed within an enclosed or partially enclosed three- dimensional space (e.g., wells, microwells or divots). In some embodiments, the substrate includes a plurality of microwells. In some embodiments, a microwell in the plurality of microwells include a feature. In some embodiments, the microwells are etched on the surface of the substrate. In some embodiments, the microwells are electroplated on the surface of the substrate. In some embodiments, the microwells are photolithographically deposited on the surface of the substrate. [0134] In some embodiments, the features of the disclosure are arrayed with EDC-coupling to the substrate. In some embodiments, the features of the disclosure are arrayed with EDC- coupling on N-hydroxysuccinimide glass slides. [0135] In some embodiments, the surface of the substrate is functionalized with silanes,
Attorney Docket No.: 057862-516001WO amines, and/or other suitable moieties. [0136] Substrates of the disclosure can be formed from a variety of solid materials, gel- based materials, colloidal materials, semi-solid materials (e.g., materials that are at least partially cross-linked), materials that are fully or partially cured (e.g., via photolithography), and materials that undergo a phase change or transition to provide physical support. Substrates of the disclosure can be insoluble in aqueous liquid. [0137] Examples of suitable substrates of the disclosure include, but are not limited to, slides (e.g., slides formed from various glasses, slides formed from various polymers), layers and/or films, membranes (e.g., porous membranes), flow cells, cuvettes, wafers (e.g., silicon), plates, or combinations thereof. [0138] In some embodiments, substrates can optionally include functional elements such as recesses, protruding structures, microfluidic elements (e.g., channels, reservoirs, electrodes, valves, seals), and various markings such a fiducials. [0139] A substrate can generally have any suitable form or format. For example, a substrate can be flat, curved, e.g., convexly or concavely curved towards the area where the interaction between the substrate and a biological sample, e.g., tissue sample, takes place. In some embodiments, a substrate is flat, e.g., planar, chip, or slide. In some embodiments, a substrate can contain one or more patterned surfaces within the substrate (e.g., channels, wells, projections, ridges, divots, hydrophobically defined spaces intra hydrophillically defined spaces, etc.). [0140] Substrates of the disclosure can be of any desired shape. For example, a substrate can be generally a thin, flat shape (e.g., a square or a rectangle). In some embodiments, a substrate structure has rounded corners (e.g., for increased safety or robustness). In some embodiments, a substrate structure has one or more cut-off corners (e.g., for use with a slide clamp or cross-table). In some embodiments, where a substrate structure is flat, the substrate structure can be any appropriate type of support having a flat surface (e.g., a chip or a slide such as a microscope slide). [0141] In some embodiments of the disclosure, substrates can optionally include various structures such as, but not limited to, projections, ridges, and channels. A substrate can be micro- patterned to limit lateral diffusion (e.g., to prevent overlap of spatial barcodes or barcoded
Attorney Docket No.: 057862-516001WO features). A substrate including such structures can be modified to allow association of analytes, features (e.g., beads), or capture probes at individual sites or regions. For example, the sites or regions where a substrate is modified with various structures can be contiguous or non- contiguous with other sites or regions. [0142] In some embodiments, the surface of a substrate can be modified so that discrete sites or regions are formed that can only have or accommodate a specific number of features, for example, a single feature. In some embodiments, the surface of a substrate can be modified so that features adhere to random sites or regions. In some embodiments, the surface of a substrate can be modified so that features are substantially regularly spaced from one another on the substrate. [0143] In some embodiments, the surface of a substrate is modified to contain one or more wells, using techniques such as (but not limited to) stamping, microetching, photlithography, or molding techniques. In some embodiments in which a substrate includes one or more wells, the substrate can be a concavity slide or cavity slide. For example, wells can be formed by one or more shallow depressions on the surface of the substrate. In some embodiments, where a substrate includes one or more wells, the wells can be formed by attaching a cassette (e.g., a cassette containing one or more chambers) to a surface of the substrate structure. [0144] In some embodiments where the substrate is modified to contain one or more structures, including but not limited to, wells, sites, spots, projections, ridges, features, or markings, the structures can include physically altered sites or regions. For example, a substrate modified with various structures can include physical properties, including, but not limited to, physical configurations, magnetic or compressive forces, chemically functionalized sites, chemically altered sites, and/or electrostatically altered sites. In some embodiments where the substrate is modified to contain various structures (e.g., wells, sites, spots, projections, ridges, features, or markings), the structures are applied in a pattern. In some embodiments, the structures can be randomly distributed. In some embodiments, the structures can be regularly spaced from one another on the substrate. In some embodiments, the structures can be substantially regularly spaced from one another on the substrate. [0145] A wide variety of different substrates can be used for the compositions and methods of the disclosure. In general, a substrate can be any suitable support material. Exemplary
Attorney Docket No.: 057862-516001WO substrates include, but are not limited to, glass, modified and/or functionalized glass, films, membranes, plastics (e.g., acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonTM, cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, and polymers, such as polystyrene, cyclic olefin copolymers (COCs), cyclic olefin polymers (COPs), polypropylene, polyethylene polycarbonate, or combinations thereof. In some embodiments, the substrates can include glass, silicon dioxide or a silicon wafer. In some embodiments, the substrates are made of glass. In some embodiments, the substrates are made of quartz or silica. In some embodiments, the substrates are transparent. In some embodiments, the substrates are translucent. [0146] In some embodiments, the substrate is a conductive substrate. Conductive substrates (e.g., electrophoretic compatible arrays) generated as described herein can be used in the spatial detection of analytes or proxies thereof. For example, an electrophoretic field can be applied to facilitate migration of analytes towards the barcoded features (e.g., features immobilized on a wafer, features or immobilized in a hydrogel film, or features immobilized on a glass slide having a conductive coating). In some embodiments, a conductive substrate can include glass (e.g., a glass slide) that has been coated with a substance or otherwise modified to confer conductive properties to the glass. Kits [0147] Further provided herein are kits for the practice of a method described herein. In some embodiments, provided herein are kits for the spatial analysis of a biological analyte or a proxy thereof in a biological sample. In some embodiments, the kits are for the spatial analysis of a nucleic acid analyte (e.g., mRNA, gDNA, rRNA, or tRNA). Such kits can include one or more substrates including the features and/or filling materials of the disclosure. In some embodiments, a kit can further include instructions for using the components of the kit to practice a method described herein. [0148] In some embodiments, the components of a kit can be in separate containers. In some other embodiments, the components of a kit can be combined in a single container. In some embodiments, the kit includes fixatives for fixing the biological sample. In some embodiments,
Attorney Docket No.: 057862-516001WO the fixatives are ethanol, methanol, acetone, formaldehyde, paraformaldehyde, Triton, glutaraldehyde, or combinations thereof. In some embodiments, the kit includes staining reagents for staining the biological sample (e.g., eosin and/or hematoxylin). In some embodiments, the kit includes a detectable label for use in staining the biological sample (e.g., a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof). [0149] The instructions for practicing the method are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, etc. The instructions can be present in the kit as a package insert, in the labeling of the container of the kit or components thereof (e.g., associated with the packaging or sub- packaging), etc. The instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, flash drive, etc. In some instances, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet), can be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate. METHODS OF THE DISCLOSURE Methods for Preparing Arrays [0150] The disclosure provides, inter alia, methods for preparing an array (e.g., hybrid microbead array), the methods include providing a substrate with a plurality of features attached to the substrate, providing a filling material, and applying the filling material to the surface of the substrate such that spaces between the plurality of features attached to the surface of the substrate substantially comprise the filling material. [0151] In an aspect, the disclosure provides methods for preparing an array, the methods include providing a substrate comprising a plurality of features attached to the substrate, wherein a feature of the plurality of features has a first refractive index at a first wavelength, providing a filling material, wherein the filling material has a second refractive index at the first wavelength, and wherein the difference between the first refractive index and the second refractive index is
Attorney Docket No.: 057862-516001WO no greater than about 10%, and applying the filling material to the surface of the substrate such that spaces between the plurality of features attached to the surface of the substrate comprise the filling material. [0152] In some embodiments, the first wavelength is a wavelength of visible light. In some exemplary embodiments, the first wavelength is in the range of about 380 to about 700 nm, or a subrange therein. [0153] A feature of the plurality of features can have a first refractive index at a first wavelength and the filling material can have a second refractive index at the first wavelength, such that the difference between the first refractive index and the second refractive index is no greater than about 10%. In some embodiments, the first refractive index and the second refractive index are different at the same wavelength. In some embodiments, the difference between the first refractive index and the second refractive index is no greater than about 10%. In some embodiments, the difference is no greater than about 1%, no greater than about 1.5%, no greater than about 2%, no greater than about 2.5%, no greater than about 3%, no greater than about 3.5%, no greater than about 4%, no greater than about 4.5%, no greater than about 5%, no greater than about 5.5%, no greater than about 6%, no greater than about 6.5%, no greater than about 7%, no greater than about 7.5%, no greater than about 8%, no greater than about 8.5%, no greater than about 9%, no greater than about 9.5%, no greater than about 10% or any values in between. [0154] In some embodiments, the first refractive index and the second refractive index are the same. In some embodiments, the fillings can be specifically selected to match the index of refraction of the features so as to reduce the refraction of light passing through the biological sample (e.g., tissue section). [0155] In an aspect, the methods of preparing an array of the disclosure can include a further step of applying a force to the filling material on the surface of the substrate and/or to the substrate such that the filling material is substantially uniformly distributed across the surface of the substrate. Any form of a suitable force can be used as appreciated by the skilled artisan. In some embodiments, the force is a mechanical force. In some embodiments, the force is a centrifugal force. In some embodiments, the centrifugal force is generated by spinning the substrate.
Attorney Docket No.: 057862-516001WO [0156] In another aspect, the methods of preparing an array of the disclosure can include a further step of subjecting the filling material on the surface of the substrate to desiccation, such that any trapped gas in the filling material is reduced or eliminated. Any methods for releasing trapped gas in the filling may be used. For example, subjecting the filling material on the surface of the substrate to shaking or vibration can be used to release trapped gas. [0157] In yet another aspect, the methods of preparing an array of the disclosure include sequencing a bead barcode within a capture probe of a feature, e.g., in order to determine the location of an analyte or a proxy thereof. The sequence of the bead barcode can be associated with the location of the bead (e.g., feature) on the substrate. In some embodiments, the sequencing is in situ sequencing. In some embodiments, the in situ sequencing is performed via sequencing-by-synthesis (SBS), sequential fluorescence hybridization, sequencing by ligation (SBL), nucleic acid hybridization, or high-throughput digital sequencing techniques. Methods for Detection of Biological Analytes in a Biological Sample [0158] The present disclosure describes methods for enhancing the sensitivity of spatial transcriptomics systems and platforms. One aspect the disclosure relates to methods for the spatial analysis of biological analytes or proxies thereof in a biological sample including use of the arrays described herein. [0159] In some embodiments, the disclosure provides methods for detecting a biological analyte in a biological sample, the methods include: (a) providing an array of the disclosure; (b) contacting the biological sample to the array, (c) imaging the biological sample to generate an image of the biological sample, (d) incubating the biological sample under conditions wherein the biological analyte binds a capture probe on a feature of the array, I determining a location of the analyte on the surface of the substrate, and (f) mapping the location of the analyte on the surface of the substrate onto a location in the biological sample using the image of the biological sample. [0160] In some embodiments, the disclosure provides methods for detecting a biological analyte in a biological sample, the methods include: (a) contacting the biological sample to an array prepared by the methods of the disclosure, (b) imaging the biological sample to generate an image of the biological sample, (c) incubating the biological sample under conditions wherein the biological analyte binds a capture probe on a feature in the plurality of features of the arrays,
Attorney Docket No.: 057862-516001WO (d) determining (i) all or a part of the sequence of the biological analyte specifically bound to the capture probe, or a complement thereof, and (ii) the sequence of the bead barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to identify the location of the analyte on the surface of the substrate, aI(e) mapping the location of the analyte on the surface of the substrate onto a location in the biological sample using the image of the biological sample. [0161] In some embodiments, the disclosure provides methods for detecting a biological analyte in a biological sample, the methods include (a) contacting the biological sample to an array prepared by the methods described herein, (b) incubating the biological sample under conditions wherein the biological analyte binds a capture probe on a feature in the plurality of features, (c) determining (i) all or a part of the sequence of the biological analyte specifically bound to the capture probe, or a complement thereof, and (ii) the sequence of the bead barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to identify the location of the analyte on the surface of the substrate. [0162] In an aspect, the methods of detecting a biological analyte of the disclosure can include sequencing a bead barcode on a capture probe of a feature in order to determine the location of an analyte or a proxy thereof. The sequence of the bead barcode can be associated with the specific location of the bead (or feature) on the substrate. In some embodiments, the sequencing is in situ sequencing. In some embodiments, the in situ sequencing is performed via sequencing-by-synthesis (SBS), sequential fluorescence hybridization, sequencing by ligation (SBL), nucleic acid hybridization, or high-throughput digital sequencing techniques. [0163] In an aspect, the methods of detecting a biological analyte of the disclosure can further include disposing or attaching the biological sample to the substrate and/or permeabilizing the biological sample to release the biological analyte therefrom. The permeabilizing can include the use of an organic solvent, a detergent, an enzyme, or a combination thereof. [0164] In some embodiments of the methods of detecting a biological analyte of the disclosure, a distortion in the image of the biological sample resulting from the feature is reduced as compared to a distortion in a reference image of the biological sample. In some embodiments, a reference image is generated using a corresponding array that does not include the filling
Attorney Docket No.: 057862-516001WO material, in particular during the imaging step. [0165] In an aspect of the methods for detecting a biological analyte of the disclosure, the biological sample can be a tissue sample, such as, fresh tissue, frozen tissue, formalin-fixed, or formalin-fixed and paraffin embedded tissue (e.g., FFPE tissue). In some embodiments, the biological sample is a tissue section, such as, a fresh and/or frozen tissue section or an FFPE tissue section. As discussed below, the biological analyte can be a nucleic acid, a mRNA, a gDNA, a rRNA, and/or a tRNA. In some embodiments, the methods further include fixing the biological sample. In some embodiments, the fixing is by using a fixative. The fixative can be any fixative including, but not limited to ethanol, methanol, acetone, formaldehyde, paraformaldehyde-Triton, glutaraldehyde, and combinations thereof. [0166] In some embodiments, the methods for detecting a biological analyte of the disclosure include staining the biological sample. Staining can be performed by using a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound or any combination thereof. In some embodiments, staining is performed by using eosin and/or hematoxylin. Spatial Analysis [0167] An exemplary workflow for the array-based spatial analysis methods disclosed herein involves the transfer of one or more analytes, or proxies thereof, from a biological sample to an array of the disclosure where a feature in the array is associated with a unique spatial location on the array. In some embodiments, the analyte (e.g., a nucleic acid) or proxy thereof hybridizes to a capture domain on the array. Subsequent analysis of the transferred analytes includes determining the identity of the analytes and the spatial location of each analyte within the biological sample. The spatial location of each analyte within the biological sample is determined based on the feature to which each analyte is bound or hybridized to on the array, and the feature’s relative spatial location within the array. [0168] In another example of a workflow, the biological analytes are proteins, in which case a set of analyte capture agents (e.g., antibody-oligonucleotide conjugates) or nucleic acid capture probes are applied to the biological sample, and the capture probes bind to the oligonucleotide of the antibody-oligonucleotide conjugates or the ligated capture probe set following hybridization and/or binding to a target of interest (e.g., protein or nucleic acids). In
Attorney Docket No.: 057862-516001WO some instances, the oligonucleotide can be cleaved from the antibody prior to being captured by the capture probe. In other instances, the oligonucleotide is not cleaved from the antibody prior to being captured by the capture probe. [0169] The spatial analysis of a biological analyte present in a biological sample may begin, for example, with imaging a fresh or frozen tissue section for histological purposes and placing the tissue section on an array containing capture probes that bind to one or more biological analytes in the sample. The tissue section is fixed and permeabilized to release analytes which can be captured by capture probes disposed adjacent to the cells of the tissue section that the analytes are released from, which allows for or facilitates downstream analytical application of the captured analytes (e.g., spatially determined gene expression or protein activity). In instances where the target analyte is a nucleic acid such as, for example mRNA, the captured nucleic acid or a nucleic acid derived therefrom (e.g., cDNA synthesized from captured RNA) is used for preparation of sequencing libraries. The libraries are sequenced, and sequencing data can be visualized, for example, to determine which genes are expressed, and where, as well as optionally, in what quantity, within a cell or tissue sample. In an example of a workflow, the cDNA generated from mRNA captured by capture probes disposed adjacent to a specific spot or region of the tissue sample generally share a common spatial barcode. Sequencing libraries are then generated from the cDNA and sequenced and the spatial barcodes are subsequently used to associate the sequence reads back to the tissue section images for spatial gene expression analysis and mapping. [0170] Another example of a workflow can include preparing a biological sample on a spatially-barcoded array. Sample preparation may include placing the sample on a slide, fixing the sample, and/or staining the biological sample for imaging. The stained sample can be imaged on the array using brightfield (to image the sample with, for example, a hematoxylin and eosin stain) and/or fluorescence (to image features comprising fluorescent moieties) modalities in such a way that positions in the spatially-barcoded array can be mapped to positions in the sample. Optionally, the sample can be destained prior to permeabilization. In some embodiments, analytes are released from the sample and capture probes forming the spatially-barcoded array hybridize to the released analytes. The analyte-bound capture probes can be analyzed to determine the identity of the analyte and where it was located on the spatial array. Where the
Attorney Docket No.: 057862-516001WO analyte is RNA (e.g., mRNA), the RNA can be reverse transcribed into cDNA containing information from the spatial barcode of the capture probe hybridized to the RNA, and an amplicon sequencing library can be prepared and sequenced to identify the RNA and where it was located on the spatial array. The mapping of positions on the spatially-barcoded array to positions in the sample can be used to provide information about the origin of the analyte in the sample. [0171] Using the methods and compositions described herein, RNA transcripts present in biological samples (e.g., tissue samples) can be used for spatial transcriptome analysis. In particular, in some cases, the barcoded oligonucleotides may be configured to prime, replicate, and consequently yield barcoded extension products from an RNA template, or derivatives thereof. For example, in some cases, the barcoded oligonucleotides may include mRNA specific priming sequences, e.g., poly-T primer segments that allow priming and replication of mRNA in a reverse transcription reaction or other targeted priming sequences. Alternatively or additionally, random RNA priming may be carried out using random N-mer primer segments of the barcoded oligonucleotides. Reverse transcriptases (RTs) can use an RNA template and a primer complementary to the 3’ end of the RNA template to direct the synthesis of the first strand complementary DNA (cDNA). Many RTs can be used in this reverse transcription reactions, including, for example, avian myeloblastosis virus (AMV) reverse transcriptase, moloney murine leukemia virus (M-MuLV or MMLV), and other variants thereof. Some recombinant M-MuLV reverse transcriptases, such as, for example, PROTOSCRIPT® II reverse transcriptase, can have reduced RNase H activity and increased thermostability when compared to their wild type counterparts, and provide higher specificity, higher yield of cDNA and more full-length cDNA products with up to 12 kilobase (kb) in length. In some embodiments, the reverse transcriptase enzyme is a mutant reverse transcriptase enzyme such as, but not limited to, mutant MMLV reverse transcriptase. In another embodiment, the reverse transcriptase is a mutant MMLV reverse transcriptase such as, but not limited to, one or more variants described in US Patent Publication No.20180312822. [0172] In a non-limiting example of the workflows described above, a biological sample (e.g., tissue section), can be fixed with methanol, stained with hematoxylin and eosin, and imaged. Optionally, the sample can be destained prior to permeabilization. The images can be
Attorney Docket No.: 057862-516001WO used to map spatial gene expression patterns back to the biological sample. A permeabilization agent (e.g., an enzyme) can be used to permeabilize the biological sample, e.g., directly on the slide. Analytes (e.g., polyadenylated mRNA) released from the overlying cells of the biological sample can be captured by capture probes within a capture area on a substrate. Reverse transcription (RT) reagents can be added to the permeabilized biological samples. Incubation with the RT reagents can produce spatially-barcoded cDNA from the captured analytes (e.g., polyadenylated mRNA). Second strand reagents (e.g., second strand primers, enzymes) can be added to the biological sample on the substrate to initiate second strand synthesis. The resulting cDNA can be denatured from the capture probe template and transferred (e.g., to a clean tube) for amplification, and/or library construction. In some embodiments, the spatially-barcoded, full- length cDNA can be amplified via PCR prior to library construction. The cDNA can be enzymatically fragmented and size-selected in order to optimize the cDNA amplicon size. P5, P7, i7, and i5 can be used as sample indexes, and TruSeq Read 2 can be added via End Repair, A-tailing, Adaptor Ligation, and PCR. The cDNA fragments can be sequenced using paired-end sequencing using TruSeq Read 1 and TruSeq Read 2 as sequencing primer sites (e.g., when utilizing Illumina based sequencers, other sequencing platforms may require different or additional sequencing primer sequences). [0173] In some embodiments, performing correlative analysis of data produced by this workflow, and other workflows described herein, can yield over 95% correlation of genes expressed across two capture areas (e.g., 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater). [0174] In some embodiments, after cDNA is generated (e.g., by reverse transcription) the cDNA can be amplified directly on the substrate surface. Generating multiple copies of the cDNA (e.g., cDNA synthesized from captured analytes) via amplification directly on the substrate surface can improve final sequencing library complexity. Thus, in some embodiments, cDNA can be amplified directly on the substrate surface by isothermal nucleic acid amplification. In some embodiments, isothermal nucleic acid amplification can amplify RNA or DNA. [0175] Any variety of staining and imaging techniques as described herein or known in the art can be used in accordance with methods described herein. In some embodiments, the staining
Attorney Docket No.: 057862-516001WO includes optical labels as described herein, including, but not limited to, fluorescent, radioactive, chemiluminescent, calorimetric, or colorimetric detectable labels. In some embodiments, the staining includes a fluorescent antibody directed to a target analyte (e.g., cell surface or intracellular proteins) in the biological sample. In some embodiments, the staining includes an immunohistochemistry stain directed to a target analyte (e.g., cell surface or intracellular proteins) in the biological sample. In some embodiments, the staining includes a chemical stain such as hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS). In some embodiments, significant time (e.g., days, months, or years) can elapse between staining and/or imaging the biological sample and performing analysis. In some embodiments, reagents for performing analysis are added to the biological sample before, contemporaneously with, or after the array is contacted to the biological sample. In some embodiments, the method may include placing the array onto the biological sample. In some embodiments, the array is a flexible array where the plurality of spatially-barcoded features (e.g., with capture probes) are attached to a flexible substrate. In some embodiments, measures are taken to slow down a reaction (e.g., cooling the temperature of the biological sample or using enzymes that preferentially perform their primary function at lower or higher temperature as compared to their optimal functional temperature) before the array is contacted with the biological sample. In some embodiments, analyzing the analyte may be without bringing the biological sample out of contact with the array. In some embodiments, analyzing the analyte is performed after the biological sample is no longer in contact with the array. In some embodiments, the biological sample is tagged with an analyte capture agent before, contemporaneously with, or after staining and/or imaging of the biological sample. In such cases, significant time (e.g., days, months, or years) can elapse between staining and/or imaging and performing analysis. In some embodiments, the array is adapted to facilitate biological analyte migration from the stained and/or imaged biological sample onto the array (e.g., using any of the materials or methods described herein). In some embodiments, a biological sample is permeabilized before being contacted with an array. In some embodiments, the rate of permeabilization is slowed prior to contacting a biological sample with an array (e.g., to limit diffusion of analytes away from their original locations in the biological sample). In some embodiments, modulating the rate of permeabilization (e.g., modulating the activity of a permeabilization reagent) can occur by modulating a condition that the biological sample is
Attorney Docket No.: 057862-516001WO exposed to (e.g., modulating temperature, pH, and/or light). In some embodiments, modulating the rate of permeabilization includes use of external stimuli (e.g., small molecules, enzymes, and/or activating reagents) to modulate the rate of permeabilization. For example, a permeabilization reagent can be provided to a biological sample prior to contact with an array, which permeabilization reagent is inactive until a condition (e.g., temperature, pH, and/or light) is changed or an external stimulus (e.g., a small molecule, an enzyme, and/or an activating reagent) is provided. Biological samples The methods of the disclosure, provided herein include detection and/or spatial analysis of a biological analyte in a biological sample. In some embodiments, a biological sample is obtained from a subject for analysis using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and/or other biological material from the subject. In some embodiments, the biological sample is a tissue sample e.g., comprising cells. In some embodiments, the biological sample (e.g., tissue sample) is a tissue microarray (TMA). A tissue microarray contains multiple representative tissue samples – which can be from different tissues or organisms – assembled on a single histologic slide. The TMA can therefore allow for high throughput analysis of multiple specimens at the same time. Tissue microarrays are paraffin blocks produced by extracting cylindrical tissue cores from different paraffin donor blocks and re-embedding these into a single recipient (microarray) block at defined array coordinates. [0176] The tissue sample can be obtained from any suitable location in a tissue or organ of a subject, e.g., a human subject. In some instances, the sample is a mouse sample. In some instances, the sample is a human sample. In some embodiments, the sample can be derived from skin, brain, breast, lung, liver, kidney, prostate, tonsil, thymus, testes, bone, lymph node, ovary, eye, heart, or spleen. [0177] In some embodiments, the tissue sample is a tissue section (e.g., tissue section obtained using a cryostat or microtome). In addition, a biological sample can be obtained from a mammal (e.g., a human), or non-mammalian organisms (e.g., a plants, an insect, an arachnid, a nematode (e.g., Caenorhabditis elegans), a fungi, an amphibian, or a fish (e.g., zebrafish)). In some embodiments, a biological sample can be obtained from a prokaryote such as a bacterium,
Attorney Docket No.: 057862-516001WO e.g., Escherichia coli, Staphylococci or Mycoplasma pneumoniae; an archaea; a virus such as Hepatitis C virus or human immunodeficiency virus; or a viroid. In some embodiments, a biological sample can be obtained from a eukaryote, such as a patient derived organoid (PDO) or patient derived xenograft (PDX). In some embodiments, a biological sample can include organoids, a miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro-anatomy. Organoids can be generated from one or more cells from a tissue, embryonic stem cells, and/or induced pluripotent stem cells, which can self- organize in three-dimensional culture owing to their self-renewal and differentiation capacities. In some embodiments, an organoid is a cerebral organoid, an intestinal organoid, a stomach organoid, a lingual organoid, a thyroid organoid, a thymic organoid, a testicular organoid, a hepatic organoid, a pancreatic organoid, an epithelial organoid, a lung organoid, a kidney organoid, a gastruloid, a cardiac organoid, or a retinal organoid. Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., cancer) or a pre-disposition to a disease, and/or individuals that are in need of therapy or suspected of needing therapy. [0178] Biological samples can include one or more diseased cells. A diseased cell can have altered metabolic properties, gene expression, protein expression, and/or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells. [0179] In some embodiments, the biological sample, e.g., the tissue sample, is fixed in a fixative including alcohol, for example methanol. In some embodiments, instead of methanol, acetone, or an acetone-methanol mixture can be used. In some embodiments, the fixation is performed after sectioning. In some embodiments, the biological sample, e.g., the tissue sample, is fixed e.g., immediately after being harvested from a subject. In any of the foregoing, the biological sample can be fixed using PAXgene. For example, the biological sample can be fixed using PAXgene in addition, or alternatively to, a fixative disclosed herein or known in the art (e.g., alcohol, acetone, acetone-alcohol, formalin, paraformaldehyde). PAXgene is a non-cross-linking mixture of different alcohols, acid and a soluble organic compound that preserves morphology and biomolecules. It is a two-reagent fixative system in which tissue is firstly fixed in a solution
Attorney Docket No.: 057862-516001WO containing methanol and acetic acid then stabilized in a solution containing ethanol. See, Ergin B. et al., J Proteome Res.2010 Oct 1;9(10):5188-96; Kap M. et al., PLoS One.; 6(11):e27704 (2011); and Mathieson W. et al., Am J Clin Pathol.; 146(1):25-40 (2016), each of which are hereby incorporated by reference in their entirety, for a description and evaluation of PAXgene for tissue fixation. [0180] In some embodiments, a biological sample can include a single analyte of interest, or more than one analyte of interest. Methods for performing multiplexed assays to analyze two or more different analytes in a single biological sample is discussed herein. [0181] A variety of steps can be performed to prepare a biological sample for analysis. Except where indicated otherwise, preparative steps can generally be combined in any manner to appropriately prepare a particular sample for analysis. [0182] In some embodiments, the biological sample can be preserved after completion of an assay with a feature or arrangement of features for additional rounds of spatial detection of analytes. In some embodiments, the biological sample, features, array, or any combination thereof can be preserved after the spatial profiling. In some embodiments, the biological sample, features, array, or combinations thereof can be protected from dehydration (e.g., drying, desiccation). In some embodiments, the biological sample, features, array, or combinations thereof, can be protected from evaporation. Methods of preserving and/or protecting biological samples, features, or arrays are known in the art. For example, in a non-limiting way, the biological sample, features, array, or combinations thereof can be covered by a reversible sealing agent. Any suitable reversible sealing agent can be used. Methods of reversible sealing are known in the art (See, e.g., WO 2019/104337, which is incorporated herein by reference). In a non-limiting way, suitable reversible sealing agents can include non-porous materials, membranes, lids, or oils (e.g., silicone oil, mineral oil). In further non-limiting examples, the biological sample, features, array, or combinations thereof can be preserved in an environmental chamber (e.g., hermetically sealed) and removed for additional rounds of spatial analysis at a later time. Biological Analytes [0183] The methods of the disclosure, provided herein include spatial analysis or detection of a biological analyte or proxy thereof in a biological sample.
Attorney Docket No.: 057862-516001WO [0184] For the purpose of this disclosure, an analyte of the disclosure will be understood to include any biological substance, structure, moiety, or component to be analyzed. [0185] Analytes can be broadly classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes. Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral coat proteins, extracellular and intracellular proteins, antibodies, and antigen binding fragments. In some embodiments, the analyte can be an organelle (e.g., nuclei or mitochondria). [0186] Cell surface features corresponding to analytes can include, but are not limited to, a receptor, an antigen, a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen-presenting complex, a major histocompatibility complex, an engineered T-cell receptor, a T-cell receptor, a B-cell receptor, a chimeric antigen receptor, an extracellular matrix protein, a posttranslational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation or lipidation) state of a cell surface protein, a gap junction, and an adherens junction. [0187] Analytes can be derived from a specific type of cell and/or a specific sub-cellular region. For example, analytes can be derived from cytosol, from cell nuclei, from mitochondria, from microsomes, and more generally, from any other compartment, organelle, or portion of a cell. Permeabilizing agents that specifically target certain cell compartments and organelles can be used to selectively release analytes from cells for analysis. [0188] Examples of nucleic acid analytes include DNA analytes such as genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and RNA/DNA hybrids. [0189] Examples of nucleic acid analytes also include RNA analytes such as various types of coding and non-coding RNA. Examples of the different types of RNA analytes include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), and viral RNA. The RNA can be a transcript (e.g., present in a tissue section). The
Attorney Docket No.: 057862-516001WO RNA can be small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA greater than 200 nucleic acid bases in length). The RNA can be double-stranded RNA or single-stranded RNA. The RNA can be circular RNA. The RNA can be a bacterial rRNA (e.g., 16S rRNA or 23S rRNA). [0190] Additional examples of analytes include mRNA and cell surface features (e.g., using the labelling agents described herein), mRNA and intracellular proteins (e.g., transcription factors), mRNA and cell methylation status, mRNA and accessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq), mRNA and metabolites (e.g., using the labelling agents described herein), a barcoded labelling agent (e.g., the oligonucleotide tagged antibodies described herein) and a V(D)J sequence of an immune cell receptor (e.g., T-cell receptor), mRNA and a perturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc finger nuclease, and/or antisense oligonucleotide as described herein). In some embodiments, a perturbation agent can be a small molecule, an antibody, a drug, an aptamer, a miRNA, a physical environmental (e.g., temperature change), or any other known perturbation agents. [0191] Analytes can include a nucleic acid molecule with a nucleic acid sequence encoding at least a portion of a V(D)J sequence of an immune cell receptor (e.g., a TCR or BCR). In some embodiments, the nucleic acid molecule is cDNA first generated from reverse transcription of the corresponding mRNA, e.g., using a poly(T) containing primer. The generated cDNA can then be barcoded using a capture probe, featuring a barcode sequence (and optionally, a UMI sequence) that hybridizes with at least a portion of the generated cDNA. Additional methods and compositions suitable for barcoding cDNA generated from mRNA transcripts including those encoding V(D)J regions of an immune cell receptor and/or barcoding methods and composition including a template switch oligonucleotide are described in PCT Patent Application PCT/US2017/057269, and PCT Patent Publication Nos. WO 2021/247568 and WO 2021/247543. V(D)J analysis can also be completed with the use of one or more labelling agents that bind to particular surface features of immune cells and associated with barcode sequences. The one or more labelling agents can include an MHC or MHC multimer. [0192] The analyte can include a nucleic acid capable of functioning as a component of a gene editing reaction, such as, for example, clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing. Accordingly, the capture probe can include a nucleic acid
Attorney Docket No.: 057862-516001WO sequence that is complementary to the analyte (e.g., a sequence that can hybridize to the CRISPR RNA (crRNA), single guide RNA (sgRNA), or an adapter sequence engineered into a crRNA or sgRNA). [0193] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. [0194] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the Applicant reserves the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific journal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art. [0195] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview of this application. [0196] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims. EXAMPLES EXAMPLE 1 Improvement of Optical Transparency [0197] This Example describes the improvement on the optical transparency when using arrays of the disclosure with filling material as opposed to arrays having no filling material. [0198] As seen from FIG.4, the optical transparency of an array including 5µm silica beads having a refractive index of 1.45 (FIG.4A), is largely improved after it is filled with polydimethylsiloxane (PDMS) having a refractive index of 1.4 (FIG.4B). EXAMPLE 2 Generalized Protocol for POC embedded microspheres
Attorney Docket No.: 057862-516001WO [0199] This is an example of a generalized protocol for POC-embedded microspheres. A polymer-coated glass microscope slide was inserted into a cassette with 9x9 mm wells. Next, 5 micron functionalized microspheres were suspended in water at 1% solids w/v and 100 microliters of which were added to a well. The wells were sealed and allowed to settle at 4° C for several hours to overnight. The slide was removed from the cassette and gently rinsed by submersion in water until excess beads were removed leaving a monolayer. The slide was allowed to dry and then was placed onto a spin coater. A PDMS mixture was prepared and degassed by a vacuum desiccator for 10 min, and 2 mL PDMS was applied to the center of the slide and the slide was spun at 6000 rpm for 2 min. PDMS was cured at 100°C for 75 minutes and allowed to cool to room temperature. Upon completion of embedding, a 10 micron thick section of fresh frozen mouse brain was placed on the slide followed by H&E staining using standard protocols. [0200] The invention can be illustrated by the following numbered paragaphs: 1. A method of preparing an array comprising: (a) providing a substrate comprising a plurality of features attached to a surface of the substrate, wherein a feature of the plurality of features has a first refractive index at a first wavelength; (b) providing a filling material, wherein the filling material has a second refractive index at the first wavelength, and wherein the difference between the first refractive index and the second refractive index is no greater than about 10%; and (c) applying the filling material to the surface of the substrate such that spaces between the plurality of features attached to the surface of the substrate substantially comprise the filling material. 2. The method of paragraph 1, further comprising (d) applying a force to the filling material on the surface of the substrate such that the filling material is substantially uniformly distributed across the surface of the substrate.
Attorney Docket No.: 057862-516001WO 3. The method of paragraph 2, wherein the force is a centrifugal or mechanical force. 4. The method of any one of paragraphs 1-3, further comprising subjecting the filling material on the surface of the substrate to desiccation, wherein an amount of trapped gas in the filling material following desiccation is less than an amount of trapped gas in the filling material prior to desiccation. 5. The method of any one of paragraphs 1-4, wherein the substrate comprises a plurality of microwells, and wherein a microwell of the plurality of microwells comprises a feature. 6. The method of paragraph 5, wherein the feature is attached to a surface of the microwell. 7. The method of paragraph 5 or 6, wherein the plurality of microwells is etched on the surface of the substrate. 8. The method of paragraph 5 or 6, wherein the plurality of microwells is electroplated on the surface of the substrate. 9. The method of paragraph 5 or 6, wherein the plurality of microwells is photolithographically deposited on the surface of the substrate. 10. The method of any one of paragraphs 1-9, wherein the surface of the substrate is functionalized with one or more silanes, amines, or a combination thereof. 11. The method of any one of paragraphs 1-10, wherein the substrate is transparent and/or translucent. 12. The method of paragraph 10, wherein the substrate is glass. 13. The method of paragraph 11, wherein the substrate is silica.
Attorney Docket No.: 057862-516001WO 14. The method of any one of paragraphs 1-13, wherein the filling material comprises a polymer, optionally wherein the polymer is in the form of a gel, a hydrogel, or a viscous liquid. 15. The method of paragraph 14, wherein the polymer is polydimethylsiloxane (PDMS). 16. The method of any one of paragraphs 1-15, wherein the filling material is reversibly attached to the substrate. 17. The method of any one of paragraphs 1-16, wherein the filling material forms a layer on the surface of the substrate comprising a thickness in the range of about 500 nanometers to about 50 micrometers. 18. The method of any one of paragraphs 1-17, wherein the filling material is in a fluid state when applied to the surface of the substrate, and wherein the method further comprises solidifying or polymerizing the filling material via heating, UV curing, or cross-linking. 19. The method of any one of paragraphs 1-18, wherein the feature is attached to the substrate via heating, gluing, UV curing, or cross-linking. 20. The method of any one of paragraphs 1-18, wherein the feature is attached to the substrate via electrostatic interactions or mechanical fixation. 21. The method of any one of paragraphs 1-20, wherein the feature is a bead. 22. The method of paragraph 21, wherein the bead has a diameter of about 0.1 µm to about 5 µm, about 1 μm to about 10 μm, about 1 μm to about 20 μm, about 1 μm to about 30 μm, about 1 μm to about 40 μm, about 1 μm to about 50 μm, about 1 μm to about 60 μm, about 1 μm to about 70 μm, about 1 μm to about 80 μm, about 1 μm to about 90 μm, about 90 μm to about 100 μm, about 80 μm to about 100 μm, about 70 μm to about 100 μm, about 60 μm to about 100 μm, about 50 μm to about 100 μm, about 40 μm to about 100 μm, about 30 μm to about 100 μm,
Attorney Docket No.: 057862-516001WO about 20 μm to about 100 μm, or about 10 μm to about 100 μm. 23. The method of any one of paragraphs 1-22, wherein the feature is composed of a material selected from the group consisting of silica, polystyrene, hydrogel, and a combination thereof. 24. The method of any one of paragraphs 1 to 23, wherein the filling material and the composition of the feature are substantially the same, and/or wherein the feature and the filling material have the same refractive index at the first wavelength. 25. The method of any one of paragraphs 1 to 23, wherein the filling material and the composition of the feature are different. 26. The method of any one of paragraphs 21 to 25 wherein the feature has a location on the substrate and comprises a plurality of polynucleotide capture probes, wherein a polynucleotide capture probe in the plurality of polynucleotide capture probes comprises a bead barcode and a capture domain, and wherein the capture domain is capable of binding to an analyte. 27. The method of paragraph 26, wherein every polynucleotide capture probe of the plurality of polynucleotide capture probes on the feature comprises the same bead barcode. 28. The method of paragraph 27, wherein for each feature of the plurality of features, the plurality of polynucleotide capture probes on the feature comprises a different bead barcode than other polynucleotide capture probes on other features of the plurality of features. 29. The method of any one of paragraphs 26 to 28, wherein the analyte is mRNA or DNA. 30. The method of any one of paragraphs 26 to 29, wherein the capture domain comprises a poly(T) sequence.
Attorney Docket No.: 057862-516001WO 31. The method of any one of paragraphs 21-30, wherein the bead barcode of the feature is associated with the location of the feature on the substrate. 32. The method of paragraph 31, further comprising determining the sequence of the bead barcode. 33. The method of paragraph 32, wherein the determining comprises sequencing, and wherein the sequencing is in situ sequencing. 34. The method of paragraph 33, wherein the in situ sequencing is performed via sequencing- by-synthesis (SBS), sequential fluorescence hybridization, sequencing by ligation, nucleic acid hybridization, or high-throughput digital sequencing techniques. 35. An array prepared by the method of any one of paragraphs 1-34. 36. A method for detecting a biological analyte in a biological sample comprising: (a) providing an array comprising a plurality of features attached to a surface of a substrate prepared by the method of any one of paragraphs 1-34; (b) contacting the biological sample with the array; (c) imaging the biological sample to generate an image of the biological sample; (d) incubating the biological sample under conditions wherein the biological analyte binds to a polynucleotide capture probe on a feature of the plurality of features; (e) determining a location of the analyte on the surface of the substrate; and (f) mapping the location of the analyte on the surface of the substrate onto a location in the biological sample using the image of the biological sample. 37. The method for detecting a biological analyte of paragraph 36, wherein determining a location of the analyte on the surface of the substrate comprises sequencing. 38. The method of paragraph 36 or 37, wherein the sequencing is in situ sequencing.
Attorney Docket No.: 057862-516001WO 39. The method of any one of paragraphs 36 to 38, wherein the in situ sequencing is performed via sequencing-by-synthesis (SBS), sequential fluorescence hybridization, sequencing by ligation, nucleic acid hybridization, or high-throughput digital sequencing techniques. 40. The method of any one of paragraphs 36 to 39, wherein a distortion in the image of the biological sample resulting from the feature is reduced as compared to a distortion in a reference image of the biological sample, and wherein the reference image is generated using a corresponding array that does not comprise the filling material during the imaging. 41. A method for detecting a biological analyte in a biological sample comprising: (a) contacting the biological sample to an array prepared by the method of any one of paragraphs 26-34; (b) imaging the biological sample to generate an image of the biological sample; (c) incubating the biological sample under conditions wherein the biological analyte binds a polynucleotide capture probe on a feature in the plurality of features; (d) determining (i) all or a part of the sequence of the biological analyte specifically bound to the polynucleotide capture probe, or a complement thereof, and (ii) the sequence of the bead barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to identify the location of the analyte on the surface of the substrate; and (e) mapping the location of the analyte on the surface of the substrate onto a location in the biological sample using the image of the biological sample. 42. The method for detecting a biological analyte of paragraph 41, wherein a distortion in the image of the biological sample resulting from the feature is reduced as compared to a distortion in a reference image of the biological sample, and wherein the reference image is generated using a corresponding spatial array that does not comprise the filling material during the imaging. 43. A method for detecting a biological analyte in a biological sample comprising:
Attorney Docket No.: 057862-516001WO (a) contacting the biological sample to an array prepared by the method of any one of paragraphs 26-34; (b) incubating the biological sample under conditions wherein the biological analyte binds a polynucleotide capture probe on a feature in the plurality of features; and (c) determining (i) all or a part of the sequence of the biological analyte specifically bound to the polynucleotide capture probe, or a complement thereof, and (ii) the sequence of the bead barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to identify the location of the analyte on the surface of the substrate. 44. The method for detecting a biological analyte of paragraph 43, wherein the biological sample comprises fresh tissue, frozen tissue, formalin fixed, or formalin-fixed, paraffin- embedded tissue. 45. The method of any one of paragraphs 36 to or 44, further comprising attaching the biological sample to the array and/or permeabilizing the biological sample to release the biological analyte therefrom, optionally wherein the permeabilizing comprises the use of an organic solvent, a detergent, an enzyme, or a combination thereof. 46. The method of any one of paragraphs 36 to 45, wherein the biological analyte is a nucleic acid. 47. The method of paragraph 46, wherein the nucleic acid is selected from the group consisting of mRNA, gDNA, rRNA, and tRNA. 48. The method of any one of paragraphs 36 to 47, wherein the method further comprises fixing the biological sample. 49. The method of paragraph 48, wherein fixing the biological sample comprises use of a fixative selected from the group consisting of: ethanol, methanol, acetone, formaldehyde, paraformaldehyde-Triton, glutaraldehyde, and combinations thereof.
Attorney Docket No.: 057862-516001WO 50. The method of any one of paragraphs 48 or 49, wherein the method further comprises staining the biological sample, imaging the biological sample, or a combination thereof. 51. The method of paragraph 50, wherein the staining comprises use of eosin and/or hematoxylin. 52. The method of paragraph 50, wherein the staining comprises use of a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof. 53. The method of any one of paragraphs 36 to 52, wherein the feature is a bead. 54. An array comprising: (a) a substrate comprising a plurality of features attached to the substrate, wherein a feature of the plurality of features has a first refractive index at a first wavelength; and (b) a filling material, wherein the filling material has a second refractive index at the first wavelength, and wherein the difference between the first refractive index and the second refractive index is no greater than about 10%, wherein the filling material substantially fills the spaces between the plurality of features attached to the surface of the substrate. 55. The array of paragraph 54, wherein the substrate further comprises a plurality of microwells, and wherein a microwell of the plurality of microwells comprises a feature of the plurality of features. 56. The array of paragraph 55, wherein the feature is attached to a surface of the microwell. 57. The array of paragraph 55 or 56, wherein the plurality of microwells is etched on the surface of the substrate.
Attorney Docket No.: 057862-516001WO 58. The array of paragraphs 55 or 56, wherein the plurality of microwells is electroplated on the surface of the substrate. 59. The array of paragraph 55 or 56, wherein the plurality of microwells is photolithographically deposited on the surface of the substrate. 60. The array of any one of paragraphs 54 to 59, wherein the surface of the substrate is functionalized with one or more silanes, amines, or a combination thereof. 61. The array of any one of paragraphs 54 to 60, wherein the substrate is transparent and/or translucent. 62. The array of paragraph 61, wherein the substrate is glass. 63. The array of paragraph 61, wherein the substrate is silica. 64. The array of any one of paragraphs 54 to 63, wherein the filling material comprises a polymer, optionally wherein the polymer is in the form of a gel, a hydrogel, or a viscous liquid. 65. The array of paragraph 64, wherein the polymer is polydimethylsiloxane (PDMS). 66. The array of any one of paragraphs 54 to 65, wherein the filling material is reversibly attached to the substrate. 67. The array of any one of paragraphs 54 to 66, wherein the filling material forms a layer on the surface of the substrate comprising a thickness in the range of about 500 nanometers to about 50 micrometers.
Attorney Docket No.: 057862-516001WO 68. The array of any one of paragraphs 54 to 67, wherein the filling material is in a fluid state when applied to the surface of the substrate, and/or wherein the method further comprises solidifying or polymerizing the filling material via heating, UV curing, or cross-linking. 69. The array of any one of paragraphs 54 to 68, wherein the feature is attached to the substrate via heating, gluing, UV curing, or cross-linking. 70. The array of any one of paragraphs 54 to 69, wherein the feature is attached to the substrate via electrostatic interactions or mechanical fixation. 71. The array of any one of paragraphs 54 to 70, wherein the feature is a bead. 72. The array of paragraph 71, wherein the bead has a diameter of about 0.1 µm to about 5 µm, about 1 μm to about 10 μm, about 1 μm to about 20 μm, about 1 μm to about 30 μm, about 1 μm to about 40 μm, about 1 μm to about 50 μm, about 1 μm to about 60 μm, about 1 μm to about 70 μm, about 1 μm to about 80 μm, about 1 μm to about 90 μm, about 90 μm to about 100 μm, about 80 μm to about 100 μm, about 70 μm to about 100 μm, about 60 μm to about 100 μm, about 50 μm to about 100 μm, about 40 μm to about 100 μm, about 30 μm to about 100 μm, about 20 μm to about 100 μm, or about 10 μm to about 100 μm. 73. The array of any one of paragraphs 54 to 72, wherein the feature is composed of a material selected from the group consisting of silica, polystyrene, hydrogel, and a combination thereof. 74. The array of paragraph 73, wherein the filling material and the composition of the feature are substantially the same, and/or wherein the feature and the filling material have the same refractive index at the first wavelength. 75. The array of paragraph 73, wherein the filling material and the composition of the feature are different.
Attorney Docket No.: 057862-516001WO 76. The array of any one of paragraphs 71 to 75, wherein the feature has a location on the substrate and comprises a plurality of polynucleotide capture probes, wherein a polynucleotide capture probe in the plurality of capture probes comprises a bead barcode and a capture domain, and wherein the capture domain is capable of binding to an analyte. 77. The array of paragraph 76, wherein every polynucleotide capture probe of the plurality of polynucleotide capture probes on the feature comprises the same bead barcode. 78. The array of paragraph 77, wherein for each feature of the plurality of features, the plurality of polynucleotide capture probes on the feature of the plurality of features comprises a different bead barcode than other polynucleotide capture probes on other features of the plurality of features. 79. The array of paragraph 76, wherein the analyte is mRNA or DNA. 80. The array of paragraph 76, wherein the capture domain comprises a poly(T) sequence. 81. The array of paragraph 76, wherein the bead barcode is associated with the specific location of the feature on the substrate. 82. A kit for the spatial analysis of a biological analyte in a biological sample comprising an array according to any one of paragraphs 35, or 54 to 81, optionally wherein the kit further comprises instructions for use thereof. 83. The kit of paragraph 82, wherein the biological analyte is a nucleic acid. 84. The kit of paragraph 83, wherein the nucleic acid is selected from the group consisting of mRNA, gDNA, rRNA, and tRNA.
Attorney Docket No.: 057862-516001WO 85. The kit of any one of paragraphs 81 to 84, wherein the kit further comprises one or more fixatives for fixing the biological sample. 86. The kit of paragraph 85, wherein the kit comprises a fixative selected from the group consisting of: ethanol, methanol, acetone, formaldehyde, paraformaldehyde-Triton, glutaraldehyde, and combinations thereof. 87. The kit of any one of paragraphs 81 to 86, wherein the kit further comprises one or more staining reagents for staining the biological sample. 88. The kit of paragraph 87, wherein the one or more staining reagents comprises eosin and/or hematoxylin. 89. The kit of paragraph 87, wherein the one or more staining reagents comprises a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof. [0201] While particular alternatives of the present disclosure have been disclosed, it is to be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. There is no intention, therefore, of limitations to the exact abstract and disclosure herein presented.
Claims
Attorney Docket No.: 057862-516001WO CLAIMS WHAT IS CLAIMED IS: 1. An array comprising: (a) a substrate comprising a plurality of features attached to the substrate, wherein a feature of the plurality of features has a first refractive index at a first wavelength; and (b) a filling material, wherein the filling material has a second refractive index at the first wavelength, and wherein the difference between the first refractive index and the second refractive index is no greater than about 10%, wherein the filling material is disposed on a surface of the substrate where the plurality of features are attached, and wherein the filling material substantially fills the spaces between the plurality of features attached to the surface of the substrate. 2. The array of claim 1, wherein the substrate further comprises a plurality of microwells, and wherein a microwell of the plurality of microwells comprises a feature of the plurality of features. 3. The array of claim 2, wherein the feature is attached to a surface of the microwell. 4. The array of claim 2 or 3, wherein the plurality of microwells is etched on the surface of the substrate. 5. The array of claims 2 or 3, wherein the plurality of microwells is electroplated on the surface of the substrate. 6. The array of claim 2 or 3, wherein the plurality of microwells is photolithographically deposited on the surface of the substrate. 7. The array of any one of claims 1 to 6, wherein the surface of the substrate is functionalized with one or more silanes, amines, or a combination thereof.
Attorney Docket No.: 057862-516001WO 8. The array of any one of claims 1 to 7, wherein the substrate is transparent and/or translucent. 9. The array of claim 8, wherein the substrate comprises glass. 10. The array of claim 8, wherein the substrate comprises silica. 11. The array of any one of claims 1 to 10, wherein the filling material comprises a polymer, optionally wherein the polymer is in the form of a gel, a hydrogel, or a viscous liquid. 12. The array of claim 11, wherein the polymer is polydimethylsiloxane (PDMS). 13. The array of any one of claims 1 to 12, wherein the filling material is reversibly attached to the substrate. 14. The array of any one of claims 1 to 13, wherein the filling material forms a layer on the surface of the substrate comprising a thickness in the range of about 500 nanometers to about 50 micrometers. 15. The array of any one of claims 1 to 14, wherein the filling material is in a fluid state when applied to the surface of the substrate, and/or wherein the filling material is solidified or polymerized via heating, UV curing, or cross-linking. 16. The array of any one of claims 1 to 15, wherein the feature is attached to the substrate via heating, gluing, UV curing, or cross-linking. 17. The array of any one of claims 1 to 16, wherein the feature is attached to the substrate via electrostatic interactions or mechanical fixation.
Attorney Docket No.: 057862-516001WO 18. The array of any one of claims 1 to 17, wherein the feature is a bead. 19. The array of claim 18, wherein the bead has a diameter of about 0.1 µm to about 5 µm, about 1 μm to about 10 μm, about 1 μm to about 20 μm, about 1 μm to about 30 μm, about 1 μm to about 40 μm, about 1 μm to about 50 μm, about 1 μm to about 60 μm, about 1 μm to about 70 μm, about 1 μm to about 80 μm, about 1 μm to about 90 μm, about 90 μm to about 100 μm, about 80 μm to about 100 μm, about 70 μm to about 100 μm, about 60 μm to about 100 μm, about 50 μm to about 100 μm, about 40 μm to about 100 μm, about 30 μm to about 100 μm, about 20 μm to about 100 μm, or about 10 μm to about 100 μm. 20. The array of any one of claims 1 to 19, wherein the feature is composed of a material selected from the group consisting of silica, polystyrene, hydrogel, and a combination thereof. 21. The array of claim 20, wherein the filling material and the composition of the feature are substantially the same, and/or wherein the feature and the filling material have the same refractive index at the first wavelength. 22. The array of claim 20, wherein the filling material and the composition of the feature are different. 23. The array of any one of claims 18 to 22, wherein the feature has a location on the substrate and comprises a plurality of polynucleotide capture probes, wherein a polynucleotide capture probe in the plurality of capture probes comprises a bead barcode and a capture domain, and wherein the capture domain is capable of binding to an analyte. 24. The array of claim 23, wherein every polynucleotide capture probe of the plurality of polynucleotide capture probes on the feature comprises the same bead barcode. 25. The array of claim 24, wherein for each feature of the plurality of features, the plurality of polynucleotide capture probes on the feature of the plurality of features comprises a different
Attorney Docket No.: 057862-516001WO bead barcode than other polynucleotide capture probes on other features of the plurality of features. 26. The array of any one of claims 23-25, wherein the analyte is mRNA or DNA. 27. The array of claim 23, wherein the capture domain comprises a poly(T) sequence, optionally wherein the poly(T) sequence comprises about 20-30 thymidine nucleotides. 28. The array of claim 23, wherein the bead barcode is associated with the specific location of the feature on the substrate. 29. A method for detecting a biological analyte in a biological sample comprising: (a) providing an array comprising (i) a substrate comprising a plurality of features attached to the substrate, wherein a feature of the plurality of features has a first refractive index at a first wavelength; and (ii) a filling material, wherein the filling material has a second refractive index at the first wavelength, and wherein the difference between the first refractive index and the second refractive index is no greater than about 10%, wherein the filling material is disposed on a surface of the substrate where the plurality of features are attached, and wherein the filling material substantially fills the spaces between the plurality of features attached to the surface of the substrate;; (b) contacting the biological sample with the array; (c) imaging the biological sample to generate an image of the biological sample; (d) incubating the biological sample under conditions wherein the biological analyte binds to a polynucleotide capture probe on a feature of the plurality of features; (e) determining a location of the analyte on the surface of the substrate; and (f) mapping the location of the analyte on the surface of the substrate onto a location in the biological sample using the image of the biological sample. 30. The method for detecting a biological analyte of claim 29, wherein determining a location
Attorney Docket No.: 057862-516001WO of the analyte on the surface of the substrate comprises sequencing. 31. The method of claim 29 or 30, wherein the sequencing is in situ sequencing. 32. The method of any one of claims 29 to 31, wherein the in situ sequencing is performed via sequencing-by-synthesis (SBS), sequential fluorescence hybridization, sequencing by ligation, nucleic acid hybridization, or high-throughput digital sequencing techniques. 33. The method of any one of claims 29 to 32, wherein a distortion in the image of the biological sample resulting from the feature is reduced as compared to a distortion in a reference image of the biological sample, and wherein the reference image is generated using a corresponding array that does not comprise the filling material during the imaging. 34. A method for detecting a biological analyte in a biological sample comprising: (a) contacting the biological sample to an array comprising (i) a substrate comprising a plurality of features attached to the substrate, wherein a feature of the plurality of features has a first refractive index at a first wavelength; and (ii) a filling material, wherein the filling material has a second refractive index at the first wavelength, and wherein the difference between the first refractive index and the second refractive index is no greater than about 10%, wherein the filling material is disposed on a surface of the substrate where the plurality of features are attached, and wherein the filling material substantially fills the spaces between the plurality of features attached to the surface of the substrate; (b) imaging the biological sample to generate an image of the biological sample; (c) incubating the biological sample under conditions wherein the biological analyte binds a polynucleotide capture probe on a feature in the plurality of features; (d) determining (i) all or a part of the sequence of the biological analyte specifically bound to the polynucleotide capture probe, or a complement thereof, and (ii) the sequence of the bead barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to identify the location of the analyte on the surface of the substrate; and
Attorney Docket No.: 057862-516001WO (e) mapping the location of the analyte on the surface of the substrate onto a location in the biological sample using the image of the biological sample. 35. The method for detecting a biological analyte of claim 34, wherein a distortion in the image of the biological sample resulting from the feature is reduced as compared to a distortion in a reference image of the biological sample, and wherein the reference image is generated using a corresponding spatial array that does not comprise the filling material during the imaging. 36. A method for detecting a biological analyte in a biological sample comprising: (a) contacting the biological sample to an array comprising (i) a substrate comprising a plurality of features attached to the substrate, wherein a feature of the plurality of features has a first refractive index at a first wavelength; and (ii) a filling material, wherein the filling material has a second refractive index at the first wavelength, and wherein the difference between the first refractive index and the second refractive index is no greater than about 10%, wherein the filling material is disposed on a surface of the substrate where the plurality of features are attached, and wherein the filling material substantially fills the spaces between the plurality of features attached to the surface of the substrate;; (b) incubating the biological sample under conditions wherein the biological analyte binds a polynucleotide capture probe on a feature in the plurality of features; and (c) determining (i) all or a part of the sequence of the biological analyte specifically bound to the polynucleotide capture probe, or a complement thereof, and (ii) the sequence of the bead barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to identify the location of the analyte on the surface of the substrate. 37. The method for detecting a biological analyte of claim 36, wherein the biological sample comprises fresh tissue, frozen tissue, formalin fixed tissue, or formalin-fixed, paraffin-embedded tissue.
Attorney Docket No.: 057862-516001WO 38. The method of any one of claims 29 to 37, further comprising attaching the biological sample to the array and/or permeabilizing the biological sample to release the biological analyte therefrom, optionally wherein the permeabilizing comprises the use of an organic solvent, a detergent, an enzyme, or a combination thereof. 39. The method of any one of claims 29 to 38, wherein the biological analyte is a nucleic acid. 40. The method of claim 39, wherein the nucleic acid is selected from the group consisting of mRNA, gDNA, rRNA, and tRNA; preferably mRNA. 41. The method of any one of claims 29 to 40, wherein the method further comprises fixing the biological sample. 42. The method of claim 41, wherein fixing the biological sample comprises use of a fixative selected from the group consisting of: ethanol, methanol, acetone, formaldehyde, paraformaldehyde-Triton, glutaraldehyde, and combinations thereof. 43. The method of any one of claims 41 or 42, wherein the method further comprises staining the biological sample, imaging the biological sample, or a combination thereof. 44. The method of claim 43, wherein the staining comprises use of eosin and/or hematoxylin. 45. The method of claim 43, wherein the staining comprises use of a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof. 46. The method of any one of claims 29 to 45 wherein the feature is a bead. 47. A method of preparing an array comprising:
Attorney Docket No.: 057862-516001WO (a) providing a substrate comprising a plurality of features attached to a surface of the substrate, wherein a feature of the plurality of features has a first refractive index at a first wavelength; (b) providing a filling material, wherein the filling material has a second refractive index at the first wavelength, and wherein the difference between the first refractive index and the second refractive index is no greater than about 10%; and (c) applying the filling material to the surface of the substrate such that spaces between the plurality of features attached to the surface of the substrate substantially comprise the filling material. 48. The method of claim 47, further comprising (d) applying a force to the filling material on the surface of the substrate and/or to the substrate such that the filling material is substantially uniformly distributed across the surface of the substrate. 49. The method of claim 48, wherein the force is a centrifugal or mechanical force. 50. The method of any one of claims 47 to 49, further comprising subjecting the filling material on the surface of the substrate to desiccation, wherein an amount of trapped gas in the filling material following desiccation is less than an amount of trapped gas in the filling material prior to desiccation. 51. The method of any one of claims 47 to 50, wherein the substrate comprises a plurality of microwells, and wherein a microwell of the plurality of microwells comprises a feature. 52. The method of claim 51, wherein the feature is attached to a surface of the microwell. 53. The method of claim 51 or 52, wherein the plurality of microwells is etched on the surface of the substrate. 54. The method of claim 51 or 52, wherein the plurality of microwells is electroplated on the
Attorney Docket No.: 057862-516001WO surface of the substrate. 55. The method of claim 51 or 52, wherein the plurality of microwells is photolithographically deposited on the surface of the substrate. 56. The method of any one of claims 47 to 55, wherein the surface of the substrate is functionalized with one or more silanes, amines, or a combination thereof. 57. The method of any one of claims 47 to 56, wherein the substrate is transparent and/or translucent. 58. The method of claim 56, wherein the substrate comprises glass. 59. The method of claim 57, wherein the substrate comprises silica. 60. The method of any one of claims 47 to 59, wherein the filling material comprises a polymer, optionally wherein the polymer is in the form of a gel, a hydrogel, or a viscous liquid. 61. The method of claim 60, wherein the polymer is polydimethylsiloxane (PDMS). 62. The method of any one of claims 47 to 61, wherein the filling material is reversibly attached to the substrate. 63. The method of any one of claims 47 to 62, wherein the filling material forms a layer on the surface of the substrate comprising a thickness in the range of about 500 nanometers to about 50 micrometers. 64. The method of any one of claims 47 to 63, wherein the filling material is in a fluid state when applied to the surface of the substrate, and wherein the method further comprises solidifying or polymerizing the filling material via heating, UV curing, or cross-linking.
Attorney Docket No.: 057862-516001WO 65. The method of any one of claims 47 to 64, wherein the feature is attached to the substrate via heating, gluing, UV curing, or cross-linking. 66. The method of any one of claims 47 to 64, wherein the feature is attached to the substrate via electrostatic interactions or mechanical fixation. 67. The method of any one of claims 47 to 66, wherein the feature is a bead. 68. The method of claim 67, wherein the bead has a diameter of about 0.1 µm to about 5 µm, about 1 μm to about 10 μm, about 1 μm to about 20 μm, about 1 μm to about 30 μm, about 1 μm to about 40 μm, about 1 μm to about 50 μm, about 1 μm to about 60 μm, about 1 μm to about 70 μm, about 1 μm to about 80 μm, about 1 μm to about 90 μm, about 90 μm to about 100 μm, about 80 μm to about 100 μm, about 70 μm to about 100 μm, about 60 μm to about 100 μm, about 50 μm to about 100 μm, about 40 μm to about 100 μm, about 30 μm to about 100 μm, about 20 μm to about 100 μm, or about 10 μm to about 100 μm. 69. The method of any one of claims 47 to 68, wherein the feature is composed of a material selected from the group consisting of silica, polystyrene, hydrogel, and a combination thereof. 70. The method of any one of claims 47 to 69, wherein the filling material and the composition of the feature are substantially the same, and/or wherein the feature and the filling material have the same refractive index at the first wavelength. 71. The method of any one of claims 47 to 69, wherein the filling material and the composition of the feature are different. 72. The method of any one of claims 67 to 71 wherein the feature has a location on the substrate and comprises a plurality of polynucleotide capture probes, wherein a polynucleotide
Attorney Docket No.: 057862-516001WO capture probe in the plurality of polynucleotide capture probes comprises a bead barcode and a capture domain, and wherein the capture domain is capable of binding to an analyte. 73. The method of claim 72, wherein every polynucleotide capture probe of the plurality of polynucleotide capture probes on the feature comprises the same bead barcode. 74. The method of claim 73, wherein for each feature of the plurality of features, the plurality of polynucleotide capture probes on the feature comprises a different bead barcode than other polynucleotide capture probes on other features of the plurality of features. 75. The method of any one of claims 72 to 74, wherein the analyte is mRNA or DNA. 76. The method of any one of claims 72 to 75, wherein the capture domain comprises a poly(T) sequence, optionally wherein the poly(T) sequence comprises about 20-30 thymidine nucleotides. 77. The method of any one of claims 67 to 76, wherein the bead barcode of the feature is associated with the location of the feature on the substrate. 78. The method of claim 77, further comprising determining the sequence of the bead barcode. 79. The method of claim 78, wherein the determining comprises sequencing, and wherein the sequencing is in situ sequencing. 80. The method of claim 79, wherein the in situ sequencing is performed via sequencing-by- synthesis (SBS), sequential fluorescence hybridization, sequencing by ligation, nucleic acid hybridization, or high-throughput digital sequencing techniques. 81. The method of any one of claims 29 to 46, further comprising extending the polynucleotide capture probe using the biological analyte as a template, thereby generating an
Attorney Docket No.: 057862-516001WO extended polynucleotide capture probe, optionally further comprising generating a complement of the extended polynucleotide capture probe. 82. The method of claim 81, wherein the determining comprises sequencing the extended polynucleotide capture probe or the complement of the extended polynucleotide capture probe. 83. An array prepared by the method of any one of claims 47 to 80. 84. A kit for the spatial analysis of a biological analyte in a biological sample comprising an array according to any one of claims 35, 54 to 80, or 83 optionally wherein the kit further comprises instructions for use thereof. 85. The kit of claim 84, wherein the biological analyte is a nucleic acid. 86. The kit of claim 85, wherein the nucleic acid is selected from the group consisting of mRNA, gDNA, rRNA, and tRNA. 87. The kit of any one of claims 84 to 86, wherein the kit further comprises one or more fixatives for fixing the biological sample. 88. The kit of claim 87, wherein the kit comprises a fixative selected from the group consisting of: ethanol, methanol, acetone, formaldehyde, paraformaldehyde-Triton, glutaraldehyde, and combinations thereof. 89. The kit of any one of claims 84 to 88, wherein the kit further comprises one or more staining reagents for staining the biological sample. 90. The kit of claim 89, wherein the one or more staining reagents comprises eosin and/or hematoxylin.
Attorney Docket No.: 057862-516001WO 91. The kit of claim 89, wherein the one or more staining reagents comprises a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.
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