CN106795650B - PF quick database building method and application thereof - Google Patents
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Abstract
The invention discloses a PF quick database building method and application thereof, wherein the PF quick database building method comprises the following steps: 1, breaking the genome DNA to obtain DNA fragments; a 2 nd step of end-repairing the DNA fragment; a 3 rd step of adding P1 and BarcodeX linker to the ends of the DNA fragment subjected to end repair; 4, performing library mixing according to the yield required by each library; 5, carrying out gap translation on the mixed library; and 6, performing quality detection on the product translated through the notch.
Description
Technical Field
The invention relates to a PF rapid database building method and application thereof, in particular to a PF rapid database building method for DNA sequencing, a CNV detection method of embryo chromosomes and a device.
Background
Pre-implantation Screening and detection (PGS) refers to a new Generation of high throughput Sequencing (NGS) method to sequence Whole Genome Amplification (WGA) products of blastomere single cells on the third day or blastocyst trophoblast cells on the fifth day by using a Next Generation of high throughput Sequencing (NGS) method In the process of culturing fertilized eggs after In vitro artificial fertilization (IVF) In vitro into blastocysts, and analyzing whether the chromosome number is abnormal and whether fragment deletion or repetition exists, so as to screen embryos with normal karyotype for implantation. Avoid abortion caused by chromosome abnormality, repeated IVF failure and pregnancy and birth of chromosome sick children.
The first test-tube baby in the world is born in 1978, and more than 500 ten thousand test-tube babies exist in the world at present; the safety of the assisted reproduction technology is fully verified. With the increasing incidence of infertility and the popularity of genetic knowledge, more and more couples are seeking to obtain normal children through assisted reproductive technology and PGS. The conventional pre-implantation genetic diagnosis technology mainly based on Fluorescence In Situ Hybridization (FISH), multiplex PCR and microarray comparative genomic Hybridization (array CGH) is disadvantageous to the popularization of PGS due to the complex operation process and large human interference factors and various defects In the aspects of detection stability, accuracy and flux. In recent years, with the rapid development of high-throughput sequencing technology, high-throughput sequencing is increasingly widely applied to the medical field; such as non-invasive prenatal Down syndrome screening based on high-throughput sequencing, HPV screening based on high-throughput sequencing, and gene diagnosis of various complex genetic diseases based on high-throughput sequencing. The common characteristics of the detection technology based on high-throughput sequencing are high automation degree, good stability, high sensitivity, large throughput and accurate result, and the high-throughput sequencing technology can be generally applied to the medical field along with the further reduction of the sequencing cost.
Disclosure of Invention
The inventors of the present invention are based on a new generation of high throughput semiconductor sequencing platform (e.g., Ion Proton)TM、Ion TorrentTMAnd the like), a PCR-Free rapid library construction technology and a (non-isometric sequence) single cell chromosome Copy Number Variation (CNV) analysis technology are independently developed, the time and the cost for preparing a sequencing library are reduced by reducing unnecessary steps, and the aneuploidy and CNV detection of the embryo single cell whole genome amplification product are realized.
The invention aims to provide a method for completing genetic detection before embryo implantation by using a high-throughput sequencing technology, and aims to solve the problem of analyzing aneuploidy or CNV of a single embryo cell sample or other single-cell-level micro samples.
The sample used in the present invention is an embryonic cell sample, a single blastomere cell or a blastocyst-stage trophoblast cell mass (generally 3 to 8). The basic operation flow is that after the cell sample is amplified by the whole genome, a sequencing library is constructed, and after the library is subjected to on-machine sequencing, sequencing data are analyzed to obtain a detection result.
In the present invention, the whole genome Amplification refers to whole genome-wide Amplification of a single cell, several cells or a trace amount of nucleic acid sample, and the method may be any of methods such as partial random Primer Amplification (hereinafter abbreviated as DOP-PCR), complete random Primer Amplification (hereinafter abbreviated as PEP-PCR), Multiple strand Displacement Amplification (hereinafter abbreviated as MDA), and Omniex PWGA. Any of commercial kits such as REPLI-g by QIAGEN, Genomeplex WGA by Sigma Aldrich, Sureplex by New England Biolabs, PicoPlex WGA by Rubicon Genomics, and Illustra Genomiphi V2 by GE Healthcare may also be used.
The invention provides a PF quick database building method, which comprises the following steps: 1, breaking the genome DNA to obtain DNA fragments; a 2 nd step of end-repairing the DNA fragment; a 3 rd step of adding P1 and BarcodeX linker to the ends of the DNA fragment subjected to end repair; 4, performing library mixing according to the yield required by each library; 5, carrying out gap translation on the mixed library; and 6, performing quality detection on the product translated through the notch.
Preferably, the genomic DNA is disrupted in step 1 with a Covaris LE220 disruptor.
Preferably, in step 2, the ends are repaired by using a polynucleotide kinase buffer, a dNTP mixed solution, T4DNA polymerase, and Klenow large fragment.
Preferably, the magnetic bead purification is performed after the 2 nd step and before the 3 rd step, after the 3 rd step and before the 4 th step, and after the 5 th step and before the 6 th step, respectively.
The second aspect of the present invention provides a CNV detection method for embryo chromosomes, comprising the following steps: constructing a library according to the PF rapid library construction method of the first aspect of the invention; performing on-machine sequencing on the constructed library to obtain a sequencing result; and carrying out information analysis on the sequencing result.
Preferably, the above-described sequencing is performed using high-throughput sequencing techniques.
Preferably, the on-machine sequencing is performed using an Ion Proton sequencer.
A third aspect of the present invention provides a CNV detection device for an embryo chromosome, comprising: a library building unit, which builds a library according to the PF quick library building method of the first aspect of the invention and outputs the library; the sequencing unit is connected to the library building unit and used for performing on-machine sequencing on the library output by the library building unit so as to output a sequencing result; and the analysis unit is connected with the sequencing unit and is used for carrying out information analysis on the sequencing result output by the sequencing unit.
Preferably, the above-described sequencing is performed using high-throughput sequencing techniques.
Preferably, the on-machine sequencing is performed using an Ion Proton sequencer.
The invention mainly develops a PCR-Free rapid library construction technology (PF library construction for short) and a single-cell CNV analysis technology (sequence with unequal length). PF library construction reduces and optimizes the steps and reaction system for constructing the library, reduces the time and cost for preparing the sequencing library, and eliminates the possibility that biased amplification possibly introduced in library PCR enrichment influences CNV judgment. The newly developed CNV analysis technology aiming at the sequences with unequal lengths can improve the utilization rate of the original sequence to the maximum extent, and can carry out express accurate CNV detection and analysis on single cell samples such as embryos and the like.
Drawings
Fig. 1 is a flowchart illustrating a library building method in the prior art.
Fig. 2 is a flowchart illustrating a PF rapid library building method of the present invention.
FIG. 3 is a schematic showing the comparative results of the disruption of WGA product and gDNA on two disruptors, respectively.
FIG. 4 is a flowchart showing the method for detecting CNV of an embryo chromosome of the present invention.
FIG. 5 is a block diagram showing the CNV detection apparatus for embryo chromosomes according to the present invention.
Detailed Description
The invention is further illustrated with reference to the following description, with reference to the accompanying drawings. It should be understood that the following embodiments are only illustrative of the present invention and are not intended to limit the scope of the present invention.
PF library construction
First, genomic DNA is prepared as a sample. 7 samples of embryos were selected, whole genome amplification was performed using the Sureplex Single Cell WGA Kit from New England Biolabs, strictly according to the Kit instructions, the amplified products were quantified for DNA, and PF library construction was performed using 100ng of DNA from each sample.
In addition, 8 samples of cell lines of known karyotypes were selected, including aneuploid samples with different deletion/duplication sizes of fragments (minimum of about 1.5 Mb). When the DNA is cultured to the optimal state, single cells are picked to simulate single cells of embryos, the whole genome amplification is also completed, DNA quantification is carried out, and 100ng of DNA is taken for each sample to complete the construction of the PF library.
Fig. 2 is a flowchart illustrating a PF rapid library building method of the present invention.
As shown in fig. 2, the advancement sample breaks. As shown in fig. 1, in the prior art, official general library manuals recommend the use of a bioraptor interrupt instrument. However, it was tested that under the guiding conditions, the disruptor was unable to disrupt the whole gene amplification product of a single cell (hereinafter referred to as WGA product) to the requirement of the 150-and 200-bp insert for sequencing. Thus, if the insert requirement for sequencing is 150-200bp, the inventors of the present invention found that the insert size requirement could be met if replaced with a Covaris LE220 disrupter, with increased throughput of disruption and significant time savings. Of course, if the insert requirement is not in the above range, a Bioruptor disruptor or other disruptor may be used.
Table 1 below shows the correlation values for two interrupters.
FIG. 3 is a schematic showing the comparative results of the disruption of WGA product and gDNA on two disruptors, respectively. Wherein lanes 1 and 10 are DNA molecular markers; lane 2 is the uninterrupted WGA product; lane 3 is uninterrupted gDNA; lanes 4 and 5 are two WGA samples disrupted with a Bioruptor disruptor; lane 6 is a gDNA sample interrupted by a Bioruptor disruptor; lanes 7 and 8 are two WGA samples interrupted by a Covaris LE220 disruptor; lane 9 is a sample of gDNA disrupted by Covaris LE220 disrupter;
when a Covaris LE220-96well plate interrupt instrument is used for sample interrupt, a transformer power supply is turned on (the input/output voltage is 220/110V), then a constant-temperature water bath switch is turned on, the temperature is set to be 4 ℃, and then the Covaris LE instrument switch and a computer are turned on; the Covaris LE water tank is taken out stably, deionized water is added into the water tank to a corresponding position, and the water tank is carefully placed into the corresponding position in the instrument; the Covaris LE on the computer and the desktop is opened, and the converter rack automatically moves downwards to the corresponding position; finally, the upper exhaust button (DEGAS PUMP) is pressed to exhaust, and the exhaust lasts at least 45 minutes.
Taking a 96-well plate, putting each hole for breaking into 2 special breaking rods, diluting 50-300 ng of the sample subjected to whole genome amplification to 80 mu L by using TE buffer solution, adding the sample into a corresponding 96-well PCR plate, sealing the membrane by using a membrane sealing machine, and performing short-time centrifugation. Pressing the green button (DOOR) of the interrupt instrument to open the DOOR, placing the 96-well PCR plate on the rack, entering the Covaris LE setting and selecting the interrupt sequence and interrupt program, and then setting the interrupt conditions.
The sample is interrupted after confirming that all settings are correct. After the interruption, the interrupted sample was slowly aspirated and stored in a 1.5 ml centrifuge tube.
Next, the DNA fragment obtained after the cleavage is subjected to end repair. 10 Xpolynucleotide kinase buffer, 10mM dNTP mixed solution, T4DNA polymerase, Klenow large fragment and T4 polynucleotide kinase are taken out from the kit stored at-20 ℃ in advance and placed on an ice box, and the reagent is well melted, mixed uniformly and centrifuged (the enzyme cannot shake).
The end repair reaction system was prepared in 2 ml centrifuge tubes in the following amounts:
80 microliter of DNA in the previous step
10 microliter of 10 Xpolynucleotide kinase buffer
dNTP mix solution (10mM) 2.5. mu.l
T4DNA polymerase 1. mu.l
Klenow Large fragment 0.1. mu.l
T4 Polynucleotide kinase 1 microliter
5.4 microliter of ultrapure water.
The total volume dispensed was 100 microliters.
The used reagents were returned to the original kit and stored at-20 ℃. Then, after shaking and centrifuging the prepared mixed solution, 20 microliters of enzyme reaction mixed solution is added for each reaction. After centrifugation with shaking, the cells were incubated in a thermostatic homogenizer (Thermomixer) at 20 ℃ for 30 minutes to complete the entire repair process.
After completion of the warm bath, preferably, 46 μ L of EB is dissolved using 1-fold volume (100 μ L) of Ampure XP beads for purification.
Next, the repaired DNA fragments are subjected to end-joining. 2 Xquick ligation buffer, Ion P1Adapter (1.25. mu.M), Ion Xpress were taken out of the kit stored at-20 ℃ in advanceTMBarcode X (1.25. mu.M) and T4DNA ligase were thawed on an ice box and centrifuged well (enzyme not shake).
The ligation reaction system was prepared in a 1.5 ml centrifuge tube using the following amounts:
44 microliter of DNA from the previous step
2X quick connect buffer 50. mu.l
Ion P1Adapter (1.25. mu.M) 1 microliter
4 microliter of T4DNA ligase (Rapid, L603-HC-L)
Ion XpressTMBarcode X (1.25. mu.M) (single plus) 1. mu.l.
The total volume dispensed was 100 microliters.
And (3) putting the used reagent back into the original kit, storing at-20 ℃, and shaking and uniformly mixing the prepared mixed solution. Ion Xpress is firstly carried outTMBarcode X (1.25. mu.M) was added to the reaction tubes in the corresponding order, and then 55. mu.l of the enzyme reaction mixture was added to each reaction tube. After centrifugation with shaking, the mixture was incubated in a homomixer (Thermomixer) at 20 ℃ for 30 minutes.
After completion of the warm bath, 20 μ L of EB is dissolved, preferably purified using 1-fold volume (100 μ L) of Ampure XP magnetic beads.
Then, library mixing is performed, and the amounts of the substances such as the single library are mixed according to the yield required for each library.
In the mixing, the final volume can be made to be 37.2. mu.l, for example.
After the library is mixed, a gap translation may be performed. The 10 XPfx buffer, the dNTP mixed solution (10mM) and MgSO were previously removed from the kit stored at-20 ℃4(50mM) and Platinum Pfx DNA polymerase (2.5U/. mu.L) were thawed on an ice box and centrifuged well (the enzyme was not shaken).
The following reaction system was prepared in a 1.5 ml centrifuge tube using the following amounts:
DNA 37.2. mu.l after mixing
5. mu.l of 10 XPfx buffer
dNTP mix solution (10mM) 5. mu.l
MgSO42 microliter (50mM)
Platinum Pfx DNA polymerase (2.5U/. mu.L) 0.8. mu.l
The total volume dispensed was 100 microliters.
The used reagent is put back into the original kit and stored at the temperature of minus 20 ℃, the prepared mixed solution is shaken and evenly mixed, and 12.8 microliters of enzyme reaction mixed solution is added in each reaction. After centrifugation with shaking, the mixture was placed in a PCR apparatus or a homomixer (Thermomixer) and reacted at 72 ℃ for 20 minutes.
After the reaction is complete, 20. mu.L of EB is dissolved, preferably purified using 1.2 volumes (60. mu.L) of Ampure XP magnetic beads.
Finally, before on-machine sequencing, quality detection is carried out on the products subjected to notch translation.
Effect
In the conventional library construction step, the concentration of the effective library can be further increased by carrying out PCR amplification on the library, and the quality of the pre-machine preparation can be improved by recovering and purifying fragments with certain sizes through subsequent gel cutting. However, the biased amplification accompanied by PCR may seriously affect the true condition of chromosome copy number variation, and the repeatability and stability of gel cutting purification are poor, and the flux is low, so that the conventional library construction scheme is not suitable for detecting a large number of samples.
The PF rapid library construction method provided by the invention optimizes the library construction steps, removes the PCR enrichment and gel cutting purification steps and carries out notch translation after mixing the libraries on the premise of ensuring that sequencing output data are basically consistent, greatly saves the input of material reagents and saves time, and the advantages are more obvious when a large number of samples are detected simultaneously.
Table 2 below shows a comparison of the sequencing output data of conventional pooling (0, 2, 5, 8 cycles of PCR).
| Sample number | Number of PCR cycles | GC content | Data utilization | Comparison rate | Repetition rate |
| S1-0 | 0 | 45.20％ | 32.22％ | 50.78％ | 27.76％ |
| S1-2 | 2 | 44.82％ | 32.28％ | 50.61％ | 27.58％ |
| S1-5 | 5 | 45.22％ | 31.27％ | 49.35％ | 28.08％ |
| S1-8 | 8 | 44.25％ | 32.39％ | 52.41％ | 29.80％ |
In the "PCR cycle number", 0 cycle is a step of not performing denaturation annealing extension, and only notch translation is performed; "GC content" is the ratio of guanine to cytosine among 4 bases in DNA. Generally, the higher the content of GC, the higher the density of DNA, and the more complex the structure; "data utilization" is the quotient of the number of unique sequences and filtered sequences. Higher values indicate higher library quality; "alignment ratio" is the ratio of the number of sequences that can be aligned to a reference sequence to the number of total sequences. Higher values indicate higher WGA product quality and library quality; the "repetition rate" is the ratio of two ends or the exact same sequence to the total number of sequences. Lower values indicate more uniform amplification of WGA and fewer amplicons introduced artificially during the pooling process.
Table 3 below compares the sequencing output data for conventional pooling (0, 8 cycles PCR) gel recovery versus no gel recovery.
Wherein, the step of building the library in 8 times of circulation in the 'rubber cutting' is the step of building the library in the conventional way, and the step of building the library in 0 time of circulation in the 'non-rubber cutting' is the step of building the library in the invention.
According to another aspect of the present invention, there is provided a CNV detection method for embryo chromosomes, as shown in fig. 4, the method comprising the steps of: constructing a library according to the PF rapid library construction method of the first aspect of the invention; performing on-machine sequencing on the constructed library to obtain a sequencing result; and carrying out information analysis on the sequencing result.
It will be appreciated by those skilled in the art that on-board sequencing can be performed by any high throughput sequencing technique known in the art, preferably using an Ion Proton sequencer, according to a specific example of the invention. The invention discovers that the sequencing result can be effectively obtained by utilizing an Ion Proton sequencer, and the method has the advantages of less sequencing time, high efficiency, accurate sequencing result and good repeatability.
According to still another aspect of the present invention, there is provided a CNV detection apparatus for an embryonic chromosome. As shown in FIG. 5, the apparatus includes a library creating unit 100, a sequencing unit 200, and an analyzing unit 300.
According to an embodiment of the present invention, the library construction unit 100 constructs and outputs a library using the above PF rapid library construction technique.
The sequencing unit 200 is connected to the library creating unit 100 and performs on-machine sequencing on the library output by the library creating unit 100 to output a sequencing result
The analysis unit 300 is connected to the sequencing unit 200 and performs information analysis on the sequencing result output by the sequencing unit 200.
It will be appreciated by those skilled in the art that any means known in the art suitable for carrying out the above operations may be employed as a component part of the various units described above. The term "coupled" as used herein is to be interpreted broadly, either directly or indirectly through an intermediary profile, the specific meaning of which is to be understood as a matter of circumstance to one of ordinary skill in the art.
Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (7)
1. A PF quick database building method is characterized by comprising the following steps:
a 1 st step of breaking the genome DNA to obtain a DNA fragment, wherein the size of the main peak of the DNA fragment is 150-200bp, and the size of the fragment is 100-250 bp;
a 2 nd step of end-repairing the DNA fragment;
a 3 rd step of adding a linker to the end of the DNA fragment subjected to end repair;
4, performing library mixing according to the yield required by each library;
5, carrying out gap translation on the mixed library; and
6, carrying out quality detection on the product translated through the notch,
wherein, the PF rapid database construction method does not comprise PCR,
and performing magnetic bead purification after the 2 nd step and before the 3 rd step, after the 3 rd step and before the 4 th step, and after the 5 th step and before the 6 th step, respectively;
and in step 1 genomic DNA is disrupted with a Covaris LE220 disruptor;
in this case, 100ng of DNA was taken for each sample to complete the PF library construction.
2. The PF fast library building method of claim 1,
in step 1 genomic DNA was subjected to 10 min breaks with a Covaris LE220 disruptor with a maximum throughput of 8.
3. The PF fast library building method of claim 1,
in the 2 nd step, the end repair is carried out by using polynucleotide kinase buffer solution, dNTP mixed solution, T4DNA polymerase and Klenow large fragment,
in step 3 the linkers are Ion P1 linker and Ion Xpress BarcodeX linker.
4. The PF fast library building method of claim 1, wherein the magnetic beads are Ampure XP magnetic beads.
5. A CNV detection device for an embryonic chromosome, comprising:
a library construction unit for constructing and outputting a library according to the PF quick library construction method of any one of claims 1 to 4;
the sequencing unit is connected to the library building unit and used for performing on-machine sequencing on the library output by the library building unit so as to output a sequencing result; and
and the analysis unit is connected with the sequencing unit and is used for carrying out information analysis on the sequencing result output by the sequencing unit.
6. The CNV detection apparatus of claim 5,
the above-machine sequencing is performed by high-throughput sequencing technology.
7. The CNV detection apparatus of claim 5,
the on-machine sequencing was performed using an Ion Proton sequencer.
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| CN113493932A (en) * | 2021-09-09 | 2021-10-12 | 北京贝瑞和康生物技术有限公司 | Method and kit for constructing capture library with high detection performance |
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| CN110945137A (en) * | 2017-09-04 | 2020-03-31 | 深圳乐土生物科技有限公司 | Detection library construction method and detection method for genetic screening before embryo implantation |
| CN109609597A (en) * | 2018-12-29 | 2019-04-12 | 北京优迅医学检验实验室有限公司 | A kind of construction method of low initial amount PCR-free high-throughput sequencing library |
| WO2021007724A1 (en) * | 2019-07-12 | 2021-01-21 | 深圳华大生命科学研究院 | Whole-genome whole-process microfluidic automatic library construction method and device |
| CN113249217A (en) * | 2021-06-30 | 2021-08-13 | 北京诺禾致源科技股份有限公司 | Automatic nucleic acid breaking method and system based on nucleic acid breaking instrument |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010064040A1 (en) * | 2008-12-02 | 2010-06-10 | Genome Research Limited | Method for use in polynucleotide sequencing |
| CN102181533A (en) * | 2011-03-17 | 2011-09-14 | 北京贝瑞和康生物技术有限公司 | Multi-sample mixed sequencing method and kit |
| WO2012083506A1 (en) * | 2010-12-24 | 2012-06-28 | 深圳华大基因科技有限公司 | Method for hla-dqb1 genotyping and related primers thereof |
| CN102690809A (en) * | 2011-03-24 | 2012-09-26 | 深圳华大基因科技有限公司 | DNA index and application thereof to construction and sequencing of mate-paired indexed library |
| CN102978206A (en) * | 2012-11-27 | 2013-03-20 | 北京诺禾致源生物信息科技有限公司 | High-throughput sequencing joint applied to hybrid library building and library building method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102864498B (en) * | 2012-09-24 | 2014-07-16 | 中国科学院天津工业生物技术研究所 | Establishment method of long mate pair library |
-
2014
- 2014-09-26 WO PCT/CN2014/087603 patent/WO2016045105A1/en active Application Filing
- 2014-09-26 CN CN201480082249.XA patent/CN106795650B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010064040A1 (en) * | 2008-12-02 | 2010-06-10 | Genome Research Limited | Method for use in polynucleotide sequencing |
| WO2012083506A1 (en) * | 2010-12-24 | 2012-06-28 | 深圳华大基因科技有限公司 | Method for hla-dqb1 genotyping and related primers thereof |
| CN102181533A (en) * | 2011-03-17 | 2011-09-14 | 北京贝瑞和康生物技术有限公司 | Multi-sample mixed sequencing method and kit |
| CN102690809A (en) * | 2011-03-24 | 2012-09-26 | 深圳华大基因科技有限公司 | DNA index and application thereof to construction and sequencing of mate-paired indexed library |
| CN102978206A (en) * | 2012-11-27 | 2013-03-20 | 北京诺禾致源生物信息科技有限公司 | High-throughput sequencing joint applied to hybrid library building and library building method thereof |
Non-Patent Citations (4)
| Title |
|---|
| KAPA Library Preparation Kit Ion Torrent(TM) Platforms;KAPA BIOSYSTEMS;《Technical Data Sheet》;20130630(第KR0573-v1.13期);第1页左栏第1段,右栏表格1附注，第5页右栏第3段 * |
| Rapid Whole-Genome Sequencing for Genetic Disease Diagnosis in Neonatal Intensive Care Units;Carol Jean Saunders et al.;《Sci. Trans. Med.》;20121003;第4卷(第154期);第13页第1段，第15页第2段 * |
| Single Cell Genomics:Advances and Future Perspectives;Macaulay I.C. et al.;《PloS Genet.》;20140131;第10卷(第1期);全文 * |
| TruSeq(R) DNA PCR-Free Sample Preparation Kit;ILLUMINA;《Data Sheet:Sequencing》;20130516;第1页第1-3段，第2页图2 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113493932A (en) * | 2021-09-09 | 2021-10-12 | 北京贝瑞和康生物技术有限公司 | Method and kit for constructing capture library with high detection performance |
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