CN112410406A - Method for determining amplification cycle number of library - Google Patents

Abstract

A method of determining the number of amplification cycles of a library, comprising: before library amplification, real-time fluorescent quantitative PCR detection is carried out on a sample to be detected in advance, a real-time fluorescent quantitative PCR detection CT value of the sample to be detected is obtained, and the cycle number required by subsequent library amplification is determined according to the CT value. The real-time fluorescence quantitative PCR detection CT value is obtained by carrying out real-time fluorescence quantitative PCR on a sample to be detected before library amplification, the cycle number of subsequent library amplification is determined according to the CT value, the problem that the library cannot be operated due to insufficient cycle number or the gene is missed to be detected due to high cycle number is effectively avoided, and quantitative reference data are provided for subsequent on-operation sequencing.

Description

Method for determining amplification cycle number of library

Technical Field

The invention relates to the technical field of gene sequencing, in particular to a method for determining the amplification cycle number of a library.

Background

In recent years, due to the rapid development of second-generation high-throughput sequencing, the sequencing cost is greatly reduced, and more researchers use the technology to deeply analyze genomes, transcriptomes and proteomes. High-throughput RNA sequencing (RNA-seq) is to perform sequencing analysis on RNA such as mRNA, LncRNA, miRNA, etc. by using a high-throughput sequencing technology. RNA-seq is capable of studying gene function and structure at a global level, explaining the molecular mechanisms in specific biological processes and disease development.

Sequencing cDNA library formed by reverse transcription of RNA in tissue or cell by high-throughput sequencing technology, calculating expression quantity of different RNAs by bioinformatics analysis, finding new transcript, positioning transcript, analyzing variable shearing, detecting fusion gene and the like

Formalin-fixed paraffin-embedded (FFPE) tissues are one of the most widely used clinical specimens, and they provide a large resource for RNA-seq, and will greatly enhance population-based cancer research. The method provides an important opportunity for further research of tumor biomarkers, the source and storage conditions of the currently received samples are difficult to control, the obtained RNA has uneven quality, the number of true amplifiable templates cannot be determined after rRNA removal and mRNA reverse transcription, and the appropriate PCR cycle number (cycle) cannot be determined, taking mRNA library construction as an example.

Disclosure of Invention

The present invention provides a method for evaluating the number of cycles of amplification products.

According to a first aspect, there is provided in one embodiment a method of determining the number of amplification cycles of a library, comprising: before library amplification, real-time fluorescent quantitative PCR detection is carried out on a sample to be detected in advance, a real-time fluorescent quantitative PCR detection CT value of the sample to be detected is obtained, and the cycle number required by subsequent library amplification is determined according to the CT value.

According to a second aspect, an embodiment provides a library construction method, including preprocessing an RNA sample to obtain a sample to be tested, performing real-time fluorescence quantitative PCR detection by the method of the first aspect to obtain a CT value for real-time fluorescence quantitative PCR detection, determining a cycle number required for subsequent library amplification according to the CT value, and performing library amplification according to the cycle number to obtain a library for on-machine sequencing.

According to the method for determining the amplification cycle number of the library in the embodiment, the real-time fluorescence quantitative PCR detection CT value is obtained by performing real-time fluorescence quantitative PCR on the sample to be detected before library amplification, and the cycle number of subsequent library amplification is determined according to the CT value, so that the problem that the library cannot be operated due to insufficient cycle number or the gene is missed to be detected due to high cycle number is effectively avoided, and quantitative reference data are provided for subsequent on-operation sequencing.

Drawings

Figure 1 shows a schematic diagram of the qPCR reaction program set-up of example 1.

Detailed Description

The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. Wherein like elements in different embodiments are numbered with like associated elements. In the following description, numerous details are set forth in order to provide a better understanding of the present application. However, those skilled in the art will readily recognize that some of the features may be omitted or replaced with other elements, materials, methods in different instances. In some instances, certain operations related to the present application have not been shown or described in detail in order to avoid obscuring the core of the present application from excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that they may be fully understood from the description in the specification and the general knowledge in the art.

Furthermore, the features, operations, or characteristics described in the specification may be combined in any suitable manner to form various embodiments. Also, the various steps or actions in the method descriptions may be transposed or transposed in order, as will be apparent to one of ordinary skill in the art. Thus, the various sequences in the specification and drawings are for the purpose of describing certain embodiments only and are not intended to imply a required sequence unless otherwise indicated where such sequence must be followed.

The numbering of the components as such, e.g., "first", "second", etc., is used herein only to distinguish the objects as described, and does not have any sequential or technical meaning. The term "connected" and "coupled" when used in this application, unless otherwise indicated, includes both direct and indirect connections (couplings).

In the prior art, the RNA sample cannot be evaluated for specific residual amount after steps of reverse transcription, double-strand synthesis and the like, so that the proper PCR cycle number (cycle) cannot be determined when a subsequent library is amplified. In the PCR amplification step of building a library, if the cycle number is low and the yield of the library is insufficient, the on-machine requirement of high-throughput sequencing cannot be met; if the number of cycles is too high, the yield of the library is too high, but only a part of the library can be taken during hybridization or machine operation, which easily results in missed detection. The total number of medical samples is usually limited, and each sample is very valuable because of its irreplaceability and no opportunity to redo it.

For RNA samples with different masses and different initial database construction quantities, in order to control the total amount of operable libraries and avoid missed detection caused by too low yield and incapability of operating the machine or too high yield, the number of cycles is strictly controlled during amplification. To address this issue, in some embodiments, the present invention adds a fluorescent quantitative PCR step prior to library amplification, directing the number of cycles of PCR amplification based on CT values, in order to precisely control library yield.

Herein, "cycle number" may also be referred to as "cycle number".

As used herein, Quantitative Real-time PCR (Quantitative Real-time PCR, qPCR) is a method for measuring the total amount of products after each Polymerase Chain Reaction (PCR) cycle in DNA amplification reaction by using fluorescent chemicals.

Herein, CT value: c represents Cycle, i.e.cycle number, T represents fluorescence threshold (threshold), and CT value means: the number of cycles that the fluorescence signal in each reaction tube passes to reach the set threshold, i.e., the number of cycles corresponding to the inflection point of exponential growth from the baseline. The fluorescence threshold is controlled within the range of the exponential growth phase of the amplification curve. The intersection of the threshold line and the amplification curve determines the CT value.

Fluorescence threshold (threshold): fluorescent signals of 15 cycles before PCR reaction are used as fluorescent background signals, and the fluorescence threshold value is 10 times of the standard deviation of the fluorescent signals of 3-15 cycles of PCR. The fluorescence threshold was set at the exponential phase of the PCR amplification.

Herein, FFPE samples (Formalin-Fixed and Parrffin-Embedded, FFPE for short) refer to samples processed by Formalin-Fixed paraffin embedding.

According to a first aspect, in some embodiments, a method of determining the number of amplification cycles of a library, comprises:

before library amplification, real-time fluorescent quantitative PCR detection is carried out on a sample to be detected in advance, a real-time fluorescent quantitative PCR detection CT value of the sample to be detected is obtained, and the cycle number required by subsequent library amplification is determined according to the CT value.

In some embodiments, the library is amplified at a cycle number of M + N, wherein M is an integer rounded off to the first decimal point of the CT number and N is-4 to 7. N can take the values of-4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, and the like.

In some embodiments, N is 0 to 7.

In some embodiments, N is 1-7.

In some embodiments, N is 0-2 if no more hybridization capture is performed on the test sample after library amplification and before machine sequencing.

In some embodiments, N is 3-7 if hybridization capture is required for the sample to be tested after library amplification and before machine sequencing.

In some embodiments, the sample to be tested is cDNA obtained by reverse transcription of an RNA sample. The sample to be tested may be a DNA sample, but since RNA cannot be evaluated for a specific residual amount after the steps of reverse transcription, double-strand synthesis, and the like, qPCR quantification is required, and thus the sample to be tested is usually cDNA obtained by reverse transcription of an RNA sample.

In some embodiments, the cDNA is double stranded.

In some embodiments, the cDNA is linker-ligated cDNA.

In some embodiments, the linker is selected from any one of an Illumina sequencing platform linker or a huada MGI sequencing platform linker, a gemin plus sequencing platform linker. The linker of the present invention is not particularly limited, and any linker that can be used in the amplification step in the second-generation sequencing library construction process can be used in the present invention.

In some embodiments, the real-time fluorescent quantitative PCR reaction system comprises at least one of DNA polymerase, magnesium ions, dntps, a fluorescent dye, a hot start reagent, a PCR enhancer, and a PCR stabilizer. Reagents required by the real-time fluorescent quantitative PCR reaction can be purchased from the market.

In some embodiments, the DNA polymerase is selected from a hot start DNA polymerase, such as Taq DNA polymerase.

In some embodiments, the fluorescent dye is selected from at least one of SYBR Green I, EvaGreen, Lc Green, SYTO, BEBO, BETO, botibo, or other fluorescent dye.

In some embodiments, the real-time fluorescent quantitative PCR reaction system contains SYBR

qPCR Master Mix、Low ROX。

In some embodiments, the real-time fluorescent quantitative PCR reaction system further comprises a primer. The same primer may be used for different samples, or different primers may be used for different samples.

In some embodiments, the primer is selected from any one of Illumina sequencing platform primer, huada gene MGI sequencing platform primer, and gieni plus sequencing platform primer.

In some embodiments, the real-time fluorescent quantitative PCR reaction conditions are as follows: at 90-98 deg.C for 30s-10 min; then 30-40 cycles are entered, each cycle is as follows: 90-98 ℃, 15-60s, 55-65 ℃, 30-60s, 70-75 ℃ and 30-60 s; after the circulation is finished, the temperature is 70-75 ℃ for 30s-10 min. Wherein, the temperature of 90-98 ℃ can specifically include but is not limited to 90 ℃, 91 ℃, 92 ℃, 93 ℃, 94 ℃, 95 ℃, 96 ℃, 97 ℃, 98 ℃ and the like; the 30s-10min specifically may include, but is not limited to, 30s, 40s, 50s, 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10min, and the like; specifically, 15-60s may include, but is not limited to, 15s, 20s, 25s, 30s, 35s, 40s, 45s, 50s, 55s, 60s, and the like; the temperature of 55-65 deg.C specifically includes but is not limited to 55 deg.C, 56 deg.C, 57 deg.C, 58 deg.C, 59 deg.C, 60 deg.C, 61 deg.C, 62 deg.C, 63 deg.C, 64 deg.C, 65 deg.C; 30-60s may specifically include, but are not limited to, 30s, 35s, 40s, 45s, 50s, 55s, 60s, and the like; the temperature of 70-75 ℃ may specifically include, but is not limited to, 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃, 75 ℃ and the like.

The specific method for RNA extraction of different samples is not particularly limited, and a method generally used by those skilled in the art can be used.

In some embodiments, the source of the RNA sample includes, but is not limited to, at least one of whole blood, fresh tissue, paraffin-embedded tissue, exfoliated cells of an organism, cell lines, stool, urine, exosomes, other bodily fluids.

In some embodiments, the organism includes, but is not limited to, at least one of an animal, a plant, a prokaryote, a protist, a microorganism, and the like. This is merely an exemplary list, and molecules carrying genetic information from a variety of organisms may be used as samples in the present invention.

In some embodiments, the animal includes, but is not limited to, a human or other animal.

In some embodiments, the microorganism includes, but is not limited to, at least one of a bacterium, a virus, a fungus, and the like.

In some embodiments, the RNA sample includes, but is not limited to, at least one of total RNA (total RNA), mRNA, LncRNA, circRNA, and the like.

Total RNA: that is, total RNA is RNA extracted and purified from a tissue and contains all RNA in a cell.

mRNA: messenger RNA is a single-stranded ribonucleic acid (dsRNA) transcribed from a DNA strand as a template and carrying genetic information that directs protein synthesis.

LncRNA: that is, Long non-coding RNA (lncRNA), is a non-coding RNA having a length of more than 200 nucleotides. lncRNA plays an important role in a plurality of life activities such as dose compensation effect (Dosage compensation effect), epigenetic regulation, cell cycle regulation, cell differentiation regulation and the like, and becomes a genetic research hotspot.

circRNA: that is, circular RNA is a special class of non-coding RNA molecules (sometimes expressed in vivo), and is also a research hotspot in the RNA field. Unlike traditional linear RNA (containing 5 'and 3' ends), the circRNA molecule is in a closed ring structure, is not influenced by RNA exonuclease, is more stable in expression and is not easy to degrade. Functionally, the circRNA molecules are rich in microRNA (miRNA) binding sites and play a role of miRNA sponge (miRNA sponge) in cells, so that the inhibition effect of miRNA on target genes of the circRNA molecules is relieved, and the expression level of the target genes is increased; this mechanism of action is known as the competitive endogenous rna (cerna) mechanism. circRNA plays an important regulatory role in disease through the interaction with disease-associated mirnas.

According to a second aspect, in some embodiments, there is provided a library construction method, including pre-treating an RNA sample to obtain a sample to be tested, performing real-time fluorescence quantitative PCR using the method of the first aspect to obtain a CT value for real-time fluorescence quantitative PCR detection of the sample to be tested, determining a cycle number required for subsequent library amplification according to the CT value, and performing library amplification according to the cycle number to obtain a library that can be used for on-machine sequencing.

In some embodiments, after library amplification, the resulting library can be applied to a sequencing platform of Illumina, DNBSEQ-T7 of Shenzhen Huada Gene science and technology Limited, Gene + Seq 2000 of Beijing Jiyin Gen plus technology Limited, Gene + Seq200, and other high throughput sequencing platforms. The sequencing platform to which the library of the present invention is applicable is not limited, and the above sequencing platforms are merely exemplary.

In some embodiments, the initial input of RNA banking samples is 5 ng-1. mu.g. The initial input amount refers to the initial input amount in the rRNA removal step. The initial input amount of RNA library building samples includes, but is not limited to, 5ng, 10ng, 20ng, 30ng, 40ng, 50ng, 60ng, 70ng, 80ng, 90ng, 100ng, 200ng, 300ng, 400ng, 500ng, 600ng, 700ng, 800ng, 900ng, 1. mu.g, and the like.

In some embodiments, if the RNA sample is total RNA, the pre-treatment comprises sequentially performing rRNA removal, disruption, reverse transcription, duplex synthesis and end-repair plus a, linker ligation, purification on the RNA sample.

rRNA generally refers to ribosomal RNA.

In some embodiments, after library amplification, the resulting product is purified and then sequenced on the machine.

In some embodiments, further comprising performing hybrid capture on the purified product prior to on-machine sequencing. The hybridization and capture step is an optional step, and the purified product can be directly subjected to on-machine sequencing without hybridization.

The hybridization capture refers to that a probe is designed and labeled by biotin, the probe can be partially or completely complementary with a target section, the target section in a sample to be detected is captured, then magnetic beads labeled with streptavidin are added into a reaction tube, a hybridization complex of the oligonucleotide probe labeled by the biotin and a DNA fragment is adsorbed by utilizing the strong non-covalent acting force between the streptavidin and the biotin, and a non-target sequence and other impurities are washed out through elution reaction, so that a target sequence capture product is obtained.

In some embodiments, if it is desired to screen for a particular RNA, e.g., mRNA, lncRNA, circRNA, etc., the RNA sample is screened prior to the disruption process.

In some embodiments, the RNA sample is first screened by at least one method including, but not limited to, poly (a) purification, rRNA removal, and the like. Methods for screening RNA samples are routine in the art.

In the following examples, the fluorescence quantifier model was used for the Qubit detection: qubit3.0 fluorescence quantifier, available from Life Technologies, Inc. (Thermo Fisher Scientific, USA).

Example 1

In this example, the reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.

This example provides a method for constructing a library of total RNA, comprising the following steps:

1. RNA extraction for Horizon FFPE fusion standards (from Horizon Discovery, UK): total RNA was extracted using the RNeasy FFPE Kit (purchased from Qiagen, Germany).

2. RNA quality control: QB was used for concentration determination, and Agilent 2100 and RNA Pico chips were used for quality control. The sample concentration was 114 ng/. mu.L, DV 20058%, where the DV200 quality index represents the percentage of RNA fragments over 200 nucleotides.

3. rRNA removal of FFPE sample RNA: the initial amount of 100ng RNA library is 6, marked as sample No. 1, sample No. 2, sample No. 3, sample No. 4, sample No. 5 and sample No. 6, and used

rRNA deletion Kit v2(Human/Mouse/Rat) and NEBNext RNA Sample Purification Beads were used to remove and purify rRNA from 6 samples, respectively, the specific steps were as follows:

3.1rRNA probe hybridization: the following single sample reagent reaction system was configured: 2 μ L

rRNA Depletion Solution、2μL

Ultra II End Prep Enzyme Mix. Reaction conditions are as follows: 2min at 95 ℃; 95-22 ℃ and 0.1 ℃/sec; 22 ℃ for 5min, and a hot lid temperature of 105 ℃.

3.2RNase H digestion: the following single sample reagent reaction system was configured: 2 μ L

RNase H Reaction Buffer、2μL

RNase H, 1. mu.L nucleic-free water. Reaction conditions are as follows: 50 ℃ for 30min, and the hot lid temperature is 55 ℃.

3.3DNase I digestion: the following single sample reagent reaction system was configured: 5. mu.L of DNase I Reaction Buffer, 2.5. mu.L of DNase I (RNase-free), 22.5. mu.L of nucleic-free water. Reaction conditions are as follows: 37 ℃ for 30min, and the temperature of the hot lid is 45 ℃.

3.4RNA purification: to the sample was added 110. mu.L (2.2X)

Incubating RNA Sample Purification Beads on ice for 15min, centrifuging the centrifugal tube for a short time, placing the centrifugal tube on a magnetic frame until the liquid is clear and transparent, and sucking and removing the supernatant; keeping the centrifuge tubes on a magnetic frame, and sequentially adding 200 mu L of 80 vol% ethanol into each centrifuge tube; repeating the steps once; after the solution is clear, sucking residual ethanol in a centrifugal tube by using a 20 mu L pipette; after the surfaces of the magnetic beads of the centrifugal tube seeds are not reflected, adding 7 mu L of NF Water (Nuclease-Free Water) into the centrifugal tube, uniformly blowing and stirring the magnetic beads by using a pipettor, and incubating for 2min at room temperature; after incubation, the tube was centrifuged briefly and placed on a magnetic rack until completely clear. Transfer the supernatant to 5. mu.L to a new tube.

4. RNA library construction: use of

The reagent in the Ultra II direct RNA Library Prep Kit for Illumina sequentially performs RNA breaking, first strand synthesis, second strand synthesis, purification, end repair and A addition and joint connection, and comprises the following specific steps:

4.1RNA disruption: the following single sample reagent reaction system was configured: 4 μ L

First Strand Synthesis Reaction Buffer、1μL Random cameras. Reaction conditions are as follows: 15min at 94 ℃.

4.2 one-chain Synthesis: the following single sample reagent reaction system was configured: 8 μ L

Strand Specificity Reagent、2μL

First Strand Synthesis Enzyme mix. Reaction conditions are as follows: 25 ℃ for 10min, 42 ℃ for 15min, 70 ℃ for 15min, and the hot lid temperature is 80 ℃.

4.3 two-chain Synthesis: the following single sample reagent reaction system was configured: 8 μ L

Second Strand Synthesis Reaction Buffer with dUTP Mix(10X)、4μL

Second Strand Synthesis Enzyme Mix, 48. mu.L of nucleic-free Water. Reaction conditions are as follows: incubate at 16 ℃ for 1 h.

4.4 purification after two-chain Synthesis: adding 144 mu L (1.8X) Axygen purified magnetic beads into the two-chain synthesis system, incubating for 10min at room temperature, centrifuging the centrifuge tube for a short time, placing the centrifuge tube on a magnetic frame until the liquid is clear and transparent, and sucking and removing the supernatant; keeping the centrifuge tubes on a magnetic frame, and sequentially adding 200 mu L of 80 vol% ethanol into each centrifuge tube; repeating the steps once; after the solution is clear, sucking residual ethanol in a centrifugal tube by using a 20 mu L pipette; after the surfaces of the magnetic beads of the centrifuge tube seeds were not reflected, 53. mu.L of 0.1 XTE was added to the centrifuge tube, wherein the TE buffer solution had the following composition: Tris-HCl 8.0 in 10mmol/L, pH, EDTA 8.0 in 1mmol/L, pH; thus, 0.1 × TE is a TE buffer diluted 10 times with NF water, and specifically, the composition of 0.1 × TE is as follows: Tris-HCl 8.0 in 1mmol/L, pH and EDTA 8.0 in 0.1mmol/L, pH. Blowing and uniformly mixing the magnetic beads by using a pipettor, and incubating for 5min at room temperature; after incubation, the tube was centrifuged briefly and placed on a magnetic rack until completely clear. The supernatant was transferred to a new tube at 50. mu.L.

4.5 end repair plus A: the following single sample reagent reaction system was configured: 7 μ L NEBNext Ultra II End Prep Reaction Buffer, 3 μ L NEBNext Ultra II End Prep Enzyme Mix. Reaction conditions are as follows: 30min at 20 ℃ and 30min at 65 ℃.

4.6 connecting joints: the linker was added singly, the linker solution was a solution of linker mother liquor diluted to a concentration of 15. mu. mol/L with TE buffer, the volume of linker solution added in this step was 1. mu.L, and the linker sequence and the preparation method of linker mother liquor were as described in example 2 of the Chinese patent application No. 202011061421.7. The following single sample reagent reaction system was configured: 30 μ L of

Ultra II Ligation Master Mix、1μL

Ligation Enhancer. Reaction conditions are as follows: incubate at 20 ℃ for 15 h.

4.7 purification after linker ligation: adding 67 mu L (0.7X) of Axygen magnetic beads into each sample, incubating for 10min at room temperature, centrifuging the centrifugal tube for a short time, placing the centrifugal tube on a magnetic frame until the liquid is clear and transparent, and absorbing and removing the supernatant; keeping the centrifuge tubes on a magnetic frame, and sequentially adding 200 mu L of 80 vol% ethanol into each centrifuge tube; repeating the steps once; after the solution is clear, sucking residual ethanol in a centrifugal tube by using a 20 mu L pipette; after the surfaces of the magnetic beads of the centrifugal tube seeds do not reflect light, adding 50 mu L of 0.1 × TE into the centrifugal tube, blowing and uniformly mixing the magnetic beads by using a pipettor, and incubating for 5min at room temperature; after incubation, the tube was centrifuged briefly and placed on a magnetic rack until completely clear. Transferring 50 mu L of the supernatant into a new tube, adding 45 mu L (0.9X) of Axygen magnetic beads, incubating at room temperature for 10min, centrifuging the centrifugal tube for a short time, placing the centrifugal tube on a magnetic frame until the liquid is clear and transparent, and sucking and removing the supernatant; keeping the centrifuge tubes on a magnetic frame, and sequentially adding 200 mu L of 80 vol% ethanol into each centrifuge tube; repeating the steps once; after the solution is clear, sucking residual ethanol in a centrifugal tube by using a 20 mu L pipette; after the surfaces of the magnetic beads of the centrifugal tube seeds do not reflect light, adding 25 mu L of 0.1 × TE into the centrifugal tube, blowing and uniformly mixing the magnetic beads by using a pipettor, and incubating for 5min at room temperature; after incubation, the tube was centrifuged briefly and placed on a magnetic rack until completely clear. In order to remove the operation difference in the library construction, after the supernatants of the 6 samples are homogenized, 2 μ L of the supernatants are taken to the qPCR quantitative reaction plate (specifically, the supernatants of the 6 samples are mixed to obtain a mixed solution, and then 2 μ L of the supernatants are taken to the qPCR quantitative reaction plate). Then transfer 20. mu.L of the supernatant mixture to 6 new PCR tubes for subsequent library amplification.

4.8qPCR quantification: using KAPA

FAST QPCR Kit Master Mix (2X) Universal, single sample reaction system as shown in Table 1.

TABLE 1

In Table 1, 1. mu.L of the primer means that the total amount of the primer added is 1. mu.L, the initial concentration of the primer is 10. mu. mol/L, and the primer is dissolved in TE buffer.

In table 1, the primer sequences are shown in the following table (i.e. the first 6 sequences in table 1 of the specification in the chinese patent application No. 202011061421.7), the primer numbers in the following table correspond to the sample numbers to be amplified one by one, and the primers in table 2 are also used for subsequent library amplification.

TABLE 2

Serial number	index1 sequence (5 '-3')	index2 sequence (5 '-3')
			1	ACCAAGCAGG(SEQ ID NO.1)	CCTGCTTGGT(SEQ ID NO.2)
2	GAGGCCTATT(SEQ ID NO.3)	AATAGGCCTC(SEQ ID NO.4)
			3	TCACCGCGCT(SEQ ID NO.5)	AGCGCGGTGA(SEQ ID NO.6)
4	TGAAGTGCAG(SEQ ID NO.7)	CTGCACTTCA(SEQ ID NO.8)
			5	CCAATGATAC(SEQ ID NO.9)	GTATCATTGG(SEQ ID NO.10)
6	ACTTCAAGCG(SEQ ID NO.11)	CGCTTGAAGT(SEQ ID NO.12)

The qPCR reaction conditions are shown in table 3 and fig. 1.

TABLE 3

The CT value after the qPCR reaction was finished was 15.98, so the value of M was 16.

5. Library amplification: the amplification reaction for each sample was as follows: 20 μ L of sample, 5 μ L of primer, 25 μ L of 2 XKAPA HiFi HotStart ReadyMix. Here, 5. mu.L of the primer means that the total amount of the primer added is 5. mu.L.

The PCR reaction conditions are shown in Table 4.

TABLE 4

In table 4, for sample nos. 1 and 2, the cycle number X is: m-4 (peak at exponential amplification), i.e., 16-4 ═ 12; for samples No. 3, 4, the cycle number X is: m +1 (midpoint of exponential amplification phase), i.e., 16+1 ═ 17; for samples No. 5, 6, the cycle number X is: m +5 (the start of the amplification plateau), i.e., 16+5 ═ 21.

6. Library purification: adding 45 mu L (0.9X) Axygen magnetic beads into each sample, incubating for 10min at room temperature, centrifuging the centrifugal tube for a short time, placing the centrifugal tube on a magnetic frame until the liquid is clear and transparent, and absorbing and removing the supernatant; keeping the centrifuge tubes on a magnetic frame, and sequentially adding 200 mu L of 80 vol% ethanol into each centrifuge tube; repeating the steps once; after the solution is clear, sucking residual ethanol in a centrifugal tube by using a 20 mu L pipette; after the surfaces of the magnetic beads of the centrifugal tube seeds do not reflect light, adding 22 mu L of TE buffer solution into the centrifugal tube, blowing and uniformly mixing the magnetic beads by using a liquid transfer device, and incubating for 5min at room temperature; after incubation, the tube was centrifuged briefly and placed on a magnetic rack until completely clear. The supernatant was transferred to a new tube at 20. mu.L.

7. Library quantification: the library was subjected to the Qubit assay, and the concentrations of sample No. 1, sample No. 2, sample No. 3, sample No. 4, sample No. 5, and sample No. 6 were 1.01 ng/. mu.L, 0.82 ng/. mu.L, 12.36 ng/. mu.L, 11.84 ng/. mu.L, 162.46 ng/. mu.L, and 171.80 ng/. mu.L, respectively.

8. Cycling the library on a machine:

the sequencer is a gigabit plus sequencer Gene + Seq-2000, the total amount of samples needing cyclization on one-time machine is 231ng, and the total volume is supplemented to 48 mu L by NF water. Therefore the average concentration of each sample should be close to 4.81 ng/. mu.L, and too low a concentration may affect other samples.

The No. 1 sample and the No. 2 sample are forced to be arranged on a computer because the concentration is too low, and the expected data size cannot be obtained, so the computer is not arranged, and the rest samples are pre-arranged on a 12G computer.

The results of the detection of the sample fusion gene are shown in Table 5.

TABLE 5

Mutation site	Sample 3	Sample 4	Sample 5	Sample 6
					EML4-ALK	√	√	/	/
CCDC6-RET	√	√	√	/
					SLC34A2-ROS1	√	√	√	√
TPM3-NTRK1	√	√	/	√
					ETV6-NTRK3	√	√	√	√

In Table 5, "√" indicates that the site was detected, and "/" indicates that the site was not detected.

From Table 5, it can be found that the libraries with cycle number M +5 have missed detection, and the libraries with cycle number M +1 have detected positions, which proves that the determination of cycle number M +1 is feasible.

Example 2

This example provides a method for constructing a library of total RNA, comprising the following steps:

1. RNA extraction of FFPE samples and tissue samples: three FFPE samples (designated sample 1, sample 2, sample 3, respectively, and all were clinical samples, sample 1 was a lymphoma sample, sample 2 was a cervical soft tissue sarcoma sample, and sample 3 was a lung cancer sample) and three tissue samples (designated sample 4, sample 5, and sample 6, respectively, sample 4 was a colorectal cancer sample, sample 5 was a breast tumor sample, and sample 6 was a lung cancer sample) were taken, and RNA was extracted using the RNeasy FFPE Kit and RNeasy Mini Kit (both purchased from Qiagen).

2. RNA quality control: the concentration of sample 1 is 102 ng/muL, DV 20024%; the concentration of sample 2 was 186 ng/. mu.L, DV 20060%; the concentration of sample 3 is 76.6 ng/. mu.L, DV 20081%; the concentration of sample 4 was 44.8 ng/. mu.L, DV 20048%; the concentration of sample 5 was 61.6 ng/. mu.L, DV 20079%; the concentration of sample 6 was 194 ng/. mu.L, DV 20090%.

3. rRNA removal of FFPE sample RNA: 500ng of each RNA sample was taken and supplemented to 12. mu.L with NF-water and used

rRNA deletion Kit v2(Human/Mouse/Rat) and

the RNA Sample Purification Beads were used to remove and purify rRNA from 6 samples, respectively, in the same manner as in step 3 of example 1.

4. RNA library construction: use of

The reagents in the Ultra II directed RNA Library Prep Kit for Illumina are sequentially subjected to RNA disruption, single-strand synthesis, double-strand synthesis, purification, end repair and A addition, linker connection and purification, and the specific method is the same as the steps 4.1 to 4.7 of the example 1. Samples 1 and 4 were interrupted at 94 ℃ for 7min, and samples 2, 3, 5, and 6 were interrupted at 94 ℃ for 15 min.

The reaction system and reaction conditions for qPCR quantification were the same as in step 4.8 of example 1. The adapters used in the adapter ligation step, the primers used in the qPCR and library amplification steps of this example were the same as in example 1.

After the qPCR reaction is completed, CT values of the sample 1, sample 2, sample 3, sample 4, sample 5, and sample 6 are 16.91, 12.89, 10.98, 11.93, 8.92, and 7.89, respectively, and therefore, when the sample 1, sample 2, sample 3, sample 4, sample 5, and sample 6 are library-amplified, corresponding M values are 17, 13, 11, 12, 9, and 8, respectively.

5. Library amplification: the amplification reaction for each sample was as follows: 20 μ L of sample, 5 μ L of primer, 25 μ L of 2 XKAPA HiFi HotStart ReadyMix.

The PCR reaction conditions were as follows: the cycle numbers of sample 1, sample 2, sample 3, sample 4, sample 5, and sample 6 were 18, 14, 12, 13, 10, and 9, respectively.

The PCR reaction procedure is shown in Table 6.

TABLE 6

6. Library purification: adding 45 mu L (0.9X) Axygen magnetic beads into each sample, incubating for 10min at room temperature, centrifuging the centrifugal tube for a short time, placing the centrifugal tube on a magnetic frame until the liquid is clear and transparent, and absorbing and removing the supernatant; keeping the centrifuge tubes on a magnetic frame, and sequentially adding 200 mu L of 80 vol% ethanol into each centrifuge tube; repeating the steps once; after the solution is clear, sucking residual ethanol in a centrifugal tube by using a 20 mu L pipette; after the surfaces of the magnetic beads of the centrifugal tube seeds do not reflect light, adding 22 mu L of TE buffer solution into the centrifugal tube, blowing and uniformly mixing the magnetic beads by using a liquid transfer device, and incubating for 5min at room temperature; after incubation, the tube was centrifuged briefly and placed on a magnetic rack until completely clear. The supernatant was transferred to 20. mu.L to a new PCR tube.

7. Library quantification: the library is subjected to the Qubit detection, the concentrations of the sample 1, the sample 2, the sample 3, the sample 4, the sample 5 and the sample 6 are respectively 13.02 ng/muL, 6.36 ng/muL, 11.36 ng/muL, 6.61 ng/muL, 7.19 ng/muL and 11.80 ng/muL, and all the samples can be subjected to on-machine sequencing without excessive PCR. The above experimental results demonstrate that the method for determining the number of amplification cycles of a library according to example 1 is versatile, and can be applied to various types of samples such as FFPE samples and tissue samples.

Example 3

This example provides a method for constructing a library of total RNA, comprising the following steps:

1. extracting RNA of the FFPE sample: three FFPE samples (designated sample 1, sample 2, and sample 3, all lung cancer samples) were taken and RNA was extracted using the RNeasy FFPE Kit (Qiagen).

2. RNA quality control: the concentration of sample 1 was 22.6 ng/. mu.L, DV 20031%; the concentration of sample 2 was 21.2 ng/. mu.L, DV 20055%; the concentration of sample 3 was 68 ng/. mu.L, DV 20072%.

3. rRNA removal of FFPE sample RNA: 100ng of each RNA sample was taken and the volume was supplemented to 12. mu.L for use

rRNA deletion Kit (Human/Mouse/Rat) and

the RNA Sample Purification Beads were used to remove and purify rRNA from 3 samples, respectively, in the same manner as in step 3 of example 1.

4. RNA library construction: use of

The reagents in the Ultra II directed RNA Library Prep Kit for Illumina are sequentially subjected to RNA disruption, single-strand synthesis, double-strand synthesis, purification, end repair and A addition, linker connection and purification, and the specific method is the same as the steps 4.1 to 4.7 of the example 1.

Wherein the joint and the primer are suitable for Illumina, and the specific sequence is shown in

Multiplex Oligos for

(Unique Dual Index UMI adapters DNA Set 1), the website of this document is shown in:

https://international.neb.com/-/media/nebus/files/manuals/manuale7395.pdfrev＝dfc680dd8df34efb9b4aede34ccc61ed&hash＝F0106827114E4DC408897A1654EF82BC。

the above primers are also used for subsequent library amplification.

Sample 1 was interrupted at 94 ℃ for 7min, and samples 2 and 3 were interrupted at 94 ℃ for 15 min.

The qPCR quantification reaction was performed as in step 4.8 of example 1.

The reaction conditions for qPCR quantification are shown in table 7.

TABLE 7

After the qPCR reaction is finished, CT values of sample 1, sample 2, and sample 3 are 12.92, 13.98, and 14.96, respectively, and corresponding M values are 13, 14, and 15, respectively.

5. Library amplification: the amplification reaction for each sample was as follows: 20 μ L of sample, 5 μ L of primer, 25 μ L of 2 XKAPA HiFi HotStart ReadyMix.

The PCR reaction conditions were as follows: the cycle numbers of sample 1, sample 2, and sample 3 were 14, 15, and 16, respectively.

The PCR reaction procedure is shown in Table 8.

TABLE 8

6. Library purification: adding 45 mu L (0.9X) Axygen magnetic beads into each sample, incubating for 10min at room temperature, centrifuging the centrifugal tube for a short time, placing the centrifugal tube on a magnetic frame until the liquid is clear and transparent, and absorbing and removing the supernatant; keeping the centrifuge tubes on a magnetic frame, and sequentially adding 200 mu L of 80 vol% ethanol into each centrifuge tube; repeating the steps once; after the solution is clear, sucking residual ethanol in a centrifugal tube by using a 20 mu L pipette; after the surfaces of the magnetic beads of the centrifugal tube seeds do not reflect light, adding 22 mu L of TE buffer solution into the centrifugal tube, blowing and uniformly mixing the magnetic beads by using a liquid transfer device, and incubating for 5min at room temperature; after incubation, the tube was centrifuged briefly and placed on a magnetic rack until completely clear. The supernatant was transferred to 20. mu.L to a new PCR tube.

7. Library quantification: the library is subjected to the Qubit detection, the concentrations of the sample 1, the sample 2 and the sample 3 are respectively 6.42 ng/muL, 12.41 ng/muL and 8.80 ng/muL, all of which can be subjected to machine sequencing without excessive PCR.

In some embodiments, by adding a real-time fluorescent quantitative PCR step, a method for controlling the ex-warehouse concentration of the library within a certain range is provided, which avoids the failure of the machine due to insufficient amplification cycles of the library and the missing detection of genes due to higher cycle number, and provides a guarantee for the subsequent process and research of the transcriptome sequencing technology (RNA-seq).

The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.

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Claims (10)

1. A method of determining the number of amplification cycles of a library, comprising: before library amplification, real-time fluorescent quantitative PCR detection is carried out on a sample to be detected in advance, a real-time fluorescent quantitative PCR detection CT value of the sample to be detected is obtained, and the cycle number required by subsequent library amplification is determined according to the CT value.

2. The method according to claim 1, wherein the library is amplified at a cycle number of M + N, wherein M is an integer rounded off from the first decimal point of the CT value, and wherein N is-4 to 7, preferably 0 to 7, and more preferably 1 to 7;

optionally, if the hybrid capture of the sample to be tested is not performed any more after the library is amplified and before the library is subjected to machine sequencing, N is 0-2;

optionally, if hybridization capture is required to be performed on the sample to be tested after library amplification and before machine sequencing, N is 3-7.

3. The method of claim 1, wherein the test sample is a cDNA obtained by reverse transcription of an RNA sample.

4. The method of claim 3, wherein the cDNA is double-stranded;

optionally, the cDNA is linker-linked cDNA.

5. The method of claim 1, wherein the real-time fluorescent quantitative PCR reaction system comprises at least one of DNA polymerase, magnesium ions, dntps, fluorescent dyes, hot start reagents, PCR enhancers, PCR stabilizers;

optionally, the DNA polymerase is selected from a hot start DNA polymerase, preferably Taq DNA polymerase;

optionally, the fluorescent dye is selected from at least one of SYBR Green I, EvaGreen, Lc Green, SYTO, BEBO, BETO, BETIBO, BOXTO;

optionally, the real-time fluorescent quantitative PCR reaction system also comprises a primer;

optionally, the real-time fluorescent quantitative PCR reaction conditions are as follows: at 90-98 deg.C for 30s-10 min; then 30-40 cycles are entered, each cycle is as follows: 90-98 ℃, 15-60s, 55-65 ℃, 30-60s, 70-75 ℃ and 30-60 s; after the circulation is finished, the temperature is 70-75 ℃ for 30s-10 min.

6. The method of claim 3, wherein the RNA sample is derived from at least one of whole blood, fresh tissue, paraffin-embedded tissue, exfoliated cells, cell lines, stool, urine, exosomes of an organism;

optionally, the organism is selected from at least one of an animal, a plant, a prokaryote, a protist, a microorganism;

optionally, the animal comprises a human, and the microorganism is at least one of a bacterium, a virus, a fungus;

optionally, the RNA sample is selected from at least one of total RNA, mRNA, LncRNA, circRNA.

7. A library construction method is characterized by comprising the steps of carrying out pretreatment on an RNA sample to obtain a sample to be tested, then carrying out real-time fluorescence quantitative PCR detection by adopting the method of any one of claims 1 to 6 to obtain a real-time fluorescence quantitative PCR detection CT value, determining the cycle number required by subsequent library amplification according to the CT value, and carrying out library amplification according to the cycle number to obtain a library for on-machine sequencing.

8. The library construction method of claim 7, wherein the initial input of the RNA sample is 5ng to 1 μ g.

9. The library construction method of claim 7, wherein if the RNA sample is total RNA, the pre-treatment comprises sequentially subjecting the RNA sample to rRNA removal, disruption, reverse transcription, double-stranded synthesis and end-repair plus A, linker ligation, purification;

optionally, after library amplification, the resulting product is purified and then subjected to on-machine sequencing.

10. The library construction method of claim 7, further comprising performing hybrid capture on the purified product prior to on-machine sequencing;

optionally, if it is desired to screen for a particular RNA, the RNA sample is screened prior to the disruption process;

optionally, the specific RNA is at least one of mRNA, LncRNA and circRNA.

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