Library building method for detecting G-quadruplex structures in oocytes, early embryos and trace cells

Abstract

The invention discloses a library building method for detecting G-quadruplex structures in oocytes, early embryos and trace cells, and belongs to the technical field of whole genome immunoprecipitation library building. The DNA G-quadruplex structure provided by the invention is a DNA secondary structure which is different from a classic double-helix structure; g-quadruplex structure abnormity can damage telomere stability, genome stability, transcriptional activity and the like; this is an important reason for poor ovum quality and embryonic development retardation. The method provided by the invention can be used for evaluating the quality of the ova of the infertile women and judging reasons causing immaturity of the ova; abnormal enrichment of in-vivo and in-vitro development arrest embryo whole genome G-quadruplex is detected and can be used as an important index for screening reasons causing embryonic development retardation; the method can be used for guiding whether corresponding clinical patients are suitable for a series of assisted reproductive technologies (ART) such as in vitro fertilization-embryo transfer (IVF-ET) and the like.

Description

Technical Field

[0001] This invention relates to the field of whole-genome immunoprecipitation library construction technology, and more specifically, to a library construction method for detecting G-quadruplex structures in oocytes, early embryos, and trace cells. Background Technology

[0002] G-quadruplexes are typically composed of a single guanine-rich nucleic acid sequence (G≥3N). x G≥3 N x G≥3 N x The secondary structure of nucleic acids (G≥3, where G refers to guanine and N refers to any base) is formed by pairing four guanine nucleotide repeat sequences and folding them into a ring with other bases in between. The four guanine nucleotides in the same plane form G-quartets by hydrogen bonds, and the G-quartets are then stacked π-π to form a G-quadruplex structure.

[0003] There are 716,310 potential G-quadruplex formation sequences in the human genome, mainly distributed in regions such as gene promoters and transcription start sites (TSS) (Varizhuk A, et al. The expanding repertoire of G4 DNA structures [J]. Biochimie, 2017, 135:54-62, Marsico G, et al, Balasubramanian S. Whole genome experimental maps of DNA G-quadruplexes in multiple species [J]. Nucleic Acids Res, 2019, 47(8):3862-3874). Nearly 50% of human genes have G-quadruplex sequences in their promoter regions (Rhodes D, et al. G-quadruplexes and their regulatory roles in biology [J]. Nucleic Acids Res, 2015, 43(18): 8627-8637). Therefore, genomic DNA... G-quadruplex structures are involved in all physiological activities of living organisms, and abnormal enrichment or deletion of G-quadruplexes in the genome can serve as one of the important markers of lesion tissues or diseased cells.

[0004] Unlike the stable DNA double helix structure, this DNA G-tetramoid structure, formed by the stacking of G-tetrads, exhibits highly dynamic variability in the genome. Each G-tetrad is composed of O atoms from each guanine. -6 Lone pairs of electrons and positively charged metal ions located at the center of the G-tetrad plane are stable, and the priority order of metal ions is: K + >Na + >NH4 + >>Li + .

[0005] Although numerous small-molecule probes and antibodies have been reported for detecting G-quadruplex structures in nucleic acids, both DNA and RNA can form G-quadruplex structures. Currently, small-molecule probes and antibodies for G-quadruplexes lack specificity for both DNA and RNA G-quadruplex structures. Therefore, accurately and sensitively detecting the dynamic changes in genomic DNA G-quadruplex structures has become urgent. Library construction and sequencing results targeting DNA G-quadruplexes can comprehensively demonstrate the enrichment and changes of G-quadruplexes in the genome, excluding interference from RNA signals. Currently, the most widely used methods for genomic DNA G-quadruplex library construction are ChIP-seq and CUT&Tag-seq techniques based on BG4 antibodies. Compared to the classic ChIP-seq technique, which uses antibodies to recognize pre-fragmented genomic fragments, the CUT&Tag-seq technique, which combines pA / G-Tn5 transposase to achieve chromatin cleavage, has the advantage of enabling in-situ target detection within cells, eliminating the damage to the in-situ G-quadruplex structure during genome fragmentation, and ensuring the accuracy of the detection results. The current problem is that, similar to ChIP-seq library preparation technology, CUT&Tag-seq library preparation technology requires a sample cell input of at least 10 cells per 10 ... 5 -10 7Only by doing so can a relatively high-quality cDNA library be obtained (Hänsel-Hertsch R, et al. Genome-wide mapping of endogenous G-quadruplex DNA structures by chromatin immunoprecipitation and high-throughput sequencing. Nat Protoc. 2018 Mar;13(3):551-564. doi: 10.1038 / nprot.2017.150. Epub 2018 Feb 22. PMID: 29470465, Li C, et al. Ligand-induced native G-quadruplex stabilization impairstranscription initiation. Genome Res. 2021 Sep;31(9):1546-1560. doi: 10.1101 / gr.275431.121. Epub 2021 Aug 16. PMID: 34400476; PMCID (PMC8415369) cannot be used to detect the G-quadruplex structure of genomic DNA in trace cells and precious samples. Summary of the Invention

[0006] The purpose of this invention is to provide a convenient and highly sensitive library preparation and sequencing method, G4tacc-seq, for detecting DNA G-quadruplexes in trace cell samples. This method addresses the limitation of current library preparation techniques in detecting G-quadruplexes in trace cell genomic DNA, enabling library preparation and sequencing of genomic DNA G-quadruplexes with an input of 50-100 cells.

[0007] The above-mentioned technical objective of this invention is achieved through the following technical solution: a library construction method for detecting G-quadruplex structures in oocytes, early embryos, and trace cells, comprising the following steps: S1. Prepare equipment and consumables, including a stereomicroscope with a heated stage, an oocyte aspiration needle, a pipette, and low-absorption centrifuge tubes; ethanol, ddH2O, AMPure magnetic beads, transposase reaction buffer, PCR amplification enzyme, PCR index primers, digitalis saponins, carrier RNA, and pA / G-Tn5 transposase. S2. Collect samples and count cells. Cells should be freshly harvested, as frozen cells cannot guarantee the good quality of the G4tacc-seq library. Cells should be intact and in a homogeneous single-cell suspension. Oocytes, early embryos, and trace cells to be tested are washed, separated, and counted using an oral pipette and placed on ice for later use. The S3, BG4, and pA / G-Tn5 complex was prepared by incubating at 95°C for 5 minutes to fully dissolve 5% digitalis saponins. 1 μL of 5% digitalis saponins was added to 1 mL of buffer 1 (B-1K) to prepare D-buffer 1 (DB-1K), and vortexed to mix. 7 μL of DB-1K, 0.5 μL of pA / G-Tn5 transposase, and 0.5 μg of BG4 antibody were added to a 200 μL low-adsorption centrifuge tube and vortexed at 4°C for 30 minutes (400 rpm). S4, prepare the cell lysis buffer. Add 50-100 cells to the wall of a 200 μL low-adsorption centrifuge tube using a pipette. To ensure the total volume of liquid and cells is <1 μL, use a pipette to aspirate excess liquid. Add 6 μL of DB-1K to the cells and incubate on ice for 10 minutes, gently vortexing once every 2.5 minutes. S5, Chromatin cleavage: Add 35 μL of DB-1K to the cell lysis buffer to ensure the stability of the DNA G-quadruplex structure during the chromatin cleavage reaction. Then add BG4 antibody and pA / G-Tn5 complex. Add 12.5 μL of pre-warmed transposase reaction buffer. Vortex at 37°C (400 rpm) for 30 minutes to perform the genome chromatin cleavage reaction. Add 2 μL of 10% SDS to the cell lysis buffer after chromatin cleavage and incubate at 55°C for 10 minutes to terminate the chromatin cleavage reaction. S6, Genomic DNA Purification: Allow AMPure magnetic beads to reach room temperature for 20 minutes before use. Add 1 μL Carrier RNA, 2 μL Internal Reference DNA (optional), and 150 μL AMPure magnetic beads to the termination reaction mixture. Mix well by pipetting and incubate at room temperature for 5 minutes. Place the low-adsorption centrifuge tube containing the termination reaction product on a magnetic rack and precipitate the magnetic bead-DNA complex for 5 minutes. Keep the low-adsorption centrifuge tube on the magnetic rack, discard the supernatant, and wash the magnetic bead-DNA complex twice with 80% ethanol. After the magnetic bead-DNA complex has air-dried, dissolve the magnetic bead-DNA complex in nuclease-free ddH2O and incubate at room temperature for 3 minutes. Place the dissolved mixture on a magnetic rack to precipitate the magnetic beads for 2 minutes. Collect the supernatant as the target genomic DNA fragment after transposition. S7, PCR amplification reaction: Prepare 50 μL of PCR reaction mixture containing PCR amplification enzyme in a 200 μL PCR tube to amplify the target DNA fragment. S8, the amplified library was purified using AMPure magnetic beads. Large fragments were filtered out using 0.4× magnetic beads, and small fragments were filtered out using 1.7× magnetic beads. The library was then eluted with 20 μL of ddH2O. S9. Library quality control and quantification: The cDNA concentration of the library was measured using a fluorescence quantitative analyzer; and the peak shape of the library was also checked. Sequencing data were compared and analyzed to quantify the enrichment of G-quadruplex DNA structures in trace amounts of early embryonic cell genomes.

[0008] Furthermore, the components of buffer 1 (B-1K) in S3 include 10 mM Tris hydrochloride at pH 7.4, 150 mM potassium chloride, 0.5 mM spermidine, and a mixture of protease inhibitors at a 1X ratio that does not contain EDTA.

[0009] Furthermore, the 50 μL PCR reaction mixture in S7 is formulated as follows: 29 μL DNA product, 10 μL 5× polymerization reaction buffer, 5 μL i5 index, 5 μL i7 index, and 1 μL polymerase.

[0010] Furthermore, in step S7, the sample is provided with a barcode Index N5XX corresponding to the i5 index and a barcode Index N7XX corresponding to the i7 index.

[0011] Furthermore, the reaction conditions for the PCR amplification reaction in S7 are as follows: Step 1: 72℃ for 3 min, 1 cycle; Step 2: 98℃ for 30 sec, 1 cycle; Step 3: 98℃ for 15 sec, 60℃ for 30 sec, 72℃ for 3 min, 16-25 cycles; Step 4: 72℃ for 5 min, 1 cycle; Step 5: Hold at 16℃.

[0012] In summary, the present invention has the following beneficial effects: 1) This invention utilizes pipettes to clean, transfer, and count cells, ensuring accurate sample cell input and minimal contamination of the library construction reaction system with operating solutions, enabling the library construction and sequencing results to sensitively and accurately reflect the true enrichment of G-quadruplexes (specifically, operation step S2).

[0013] 2) In this invention, fresh cells are harvested for cDNA library construction targeting the G-quadruplex structure. To minimize the impact of cellular stress caused by in vitro operations on the genomic DNA G-quadruplex library construction, the EP tubes containing the cells to be tested should be immediately placed on ice to pause cellular physiological activity (specifically, step S2). Furthermore, to ensure that the genomic DNA G-quadruplex structure is not damaged, a lysis buffer DB-1K is prepared using KCl, placing the genomic DNA G-quadruplex structure in a KCl-containing solution. + It remains stable in the in vitro environment.

[0014] 3) Unlike traditional ChIP-seq and CUT&Tag library construction technologies, the reaction system for obtaining the target genomic DNA fragments in this invention is a single-tube operation (specifically operation steps S2-6), which shortens the operation time and reduces the loss of the target genomic DNA fragments caused by frequent pipetting.

[0015] 4) In this invention, AMPure magnetic beads are used to purify the genomic target DNA fragment after chromatin cleavage reaction (specifically, operation step S6). This method differs from the traditional phenol-chloroform nucleic acid precipitation method, making it more suitable for obtaining trace amounts of DNA fragments and reducing the loss of genomic target DNA fragments.

[0016] 5) This invention proposes to establish a map of the distribution and enrichment of G4 strand structures in whole-genome DNA in trace oocytes and early embryos using G4tacc-seq.

[0017] 6) This invention proposes that disrupting the dynamic changes in the DNA G-quadruplex structure during the maternal-to-zygotic transition (MZT) can cause developmental arrest in the 1-cell of early mouse embryos and lead to a large accumulation of genomic DNA damage. Figure 1 As shown in the figure, 1-Cell developmental arrest can be considered as one of the phenotypes of genomic DNA structural dysregulation.

[0018] 7) This invention also relates to screening for the causes of early embryonic MZT failure and developmental arrest. Later, by detecting abnormal enrichment or deletion of in situ G-quadruplexes in the genome of developmentally arrested fertilized eggs (1-Cell developmental arrest), it can be determined whether the patient has pathogenic factors related to abnormalities in nucleic acid helicases and topoisomerases. Abnormalities in nucleic acid structure regulation-related enzymes in patients manifest as infertility, characterized by abnormal accumulation of DNA damage in fertilized eggs, resulting in embryonic developmental arrest and inability to conceive. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating how disrupting the dynamic changes of DNA G-quadruplexes in mouse zygotes leads to embryonic developmental arrest at the 1-Cell stage. Figure 2 This image shows the peak diagram of the library used to construct a cDNA library of G-quadruplex genomic DNA from the 4-Cell stage of early embryos via G4tacc-seq. It illustrates how the BG4 antibody specifically enriched for the target DNA fragment in the G4tacc-seq library construction system, compared to the group without antibody.

[0020] Figure 3To construct cDNA libraries of G-quadruplex DNA from oocytes, early embryos, and trace amounts of HeLa cells using G4tacc-seq, a distribution map of the genomic signal of G-quadruplex DNA was generated. Detailed Implementation

[0021] This invention proposes a library preparation method for detecting DNA G-quadruplex structures in the genomes of oocytes, early embryos, and trace cells, aiming to address the current lack of sensitive library preparation kits for detecting DNA G-quadruplex structures in trace cells.

[0022] The present invention will be further illustrated below with specific examples. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0023] In this embodiment, TruePrep Amplify Enzyme was selected as the PCR amplification enzyme, and TruePrepIndex Kit V2 for Illumina was selected to construct the library. The transposase reaction buffer can be a commercially available Tn5 transposase buffer commonly used in this field, such as the TTBL component of the TruePrep DNA library Prep Kit V2 for Illumina kit from Nanjing Novizan Biotechnology Co., Ltd., namely TruePrep Tagment Buffer L.

[0024] In this embodiment, the method for detecting the distribution and enrichment of DNA G-quadruplexes in the genomes of 25 4-cell embryos using G4tacc-seq library preparation and sequencing technology includes: 4-Cell Embryo Acquisition and Counting: Four-week-old female mice were selected and injected with 7 units of PMSG (pregnant mareserum gonadotropin, Ningbo Sansheng Pharmaceutical Co., Ltd.) to mimic the in vivo effect of FSH and promote follicle development. 44-48 hours after PMSG administration, 7 units of hCG (Human Chorionic Gonadotropin, Ningbo Sansheng Pharmaceutical Co., Ltd.) were injected. The hCG-injected female mice were then mated with 10-12 week-old male mice. Twenty hours after hCG injection, the oviduct was removed from the mated female mice by cervical dissection, and the enlarged portion of the oviduct was placed in M2 (Sigma) culture medium. Then, in M2 culture medium, the enlarged portion of the oviduct was punctured with a 1 ml syringe needle to release the fertilized egg. Fertilized eggs were collected and repeatedly pipetted using a pipette 1.5 times the diameter of the fertilized egg until the cumulus cells surrounding the fertilized egg were removed. The collected eggs were washed with clean M2 culture medium and then placed in KSOM culture drops for further culture. The ratio of fertilized eggs to KSOM culture medium was approximately 1 fertilized egg per 2 μL of culture medium, and mineral oil was added to form a liquid seal to maintain long-term stability of the culture medium. The fertilized eggs were cultured at a constant temperature of 37°C in a 5% high-purity CO2 incubator. Twenty-five early 4-cell mouse embryos were obtained 68 hours after hCG injection, washed three times with 0.2% BSA / PBS, and placed on ice for later use.

[0025] Preparation of BG4 antibody and pA / G-Tn5 complex: Dissolve 5% digitalis saponin by incubation at 95°C for 5 minutes. Add 1 μL of 5% digitalis saponin to 1 mL of buffer 1 (B-1K) to prepare D-buffer 1 (DB-1K), and vortex to mix. Add 7 μL of DB-1K, 0.5 μL of pA / G-Tn5, and 0.5 μg of antibody to a 200 μL low-adsorption centrifuge tube and vortex at 4°C for 30 minutes (400 rpm).

[0026] Cell lysis buffer preparation: Add 25 embryos to a 200 μL low-absorption centrifuge tube (total volume of liquid and cells <1 μL). Add 6 μL of DB-1K to the cells and incubate on ice for 10 minutes, gently vortexing once every 2.5 minutes.

[0027] Chromatin cleavage: Add 35 μL of DB-1K to the cell lysis buffer, followed by the BG4 antibody and pA / G-Tn5 mixture. Add 12.5 μL of pre-warmed transposase reaction buffer. Vortex at 37°C (400 rpm) for 30 minutes using a thermal mixer (Topheer mixer). Add 2 μL of 10% SDS to the chromatin-cleaved cell lysis buffer and incubate at 55°C for 10 minutes to terminate the chromatin cleavage reaction.

[0028] Genomic DNA purification: Allow AMPure magnetic beads to reach room temperature for 20 minutes before use. Add 1 μL of carrier RNA, 2 μL of internal control DNA (optional), and 150 μL of AMPure magnetic beads to the termination reaction mixture. Mix well by pipetting and incubate at room temperature for 5 minutes. Place the low-adsorption centrifuge tube containing the termination reaction product on a magnetic rack (Life Technologies) to precipitate the magnetic bead-DNA complex for 5 minutes. Keep the EP tube on the magnetic rack, discard the supernatant, and wash the magnetic bead-DNA complex twice with 80% ethanol. After the magnetic bead-DNA complex has air-dried, dissolve the magnetic bead-DNA complex in nuclease-free ddH2O and incubate at room temperature for 3 minutes. Place the solution on a magnetic rack to precipitate the magnetic beads for 2 minutes. Collect the supernatant as the transposable genomic DNA.

[0029] PCR amplification reaction: Prepare 50 μL of PCR reaction mixture containing PCR amplification enzyme (specifically, Vazyme, TruePrep Amplify Enzyme) in a 0.2 mL PCR tube to amplify the transposable DNA fragment; The samples are marked with barcodes corresponding to the i5 index (Index N5XX) and the i7 index (Index N7XX) to facilitate subsequent mixing, sequencing, and data splitting of different samples.

[0030] cDNA library purification: Large fragments were filtered out using 0.4× magnetic beads. Small fragments were filtered out using 1.7× magnetic beads. The library was purified by elution with 20 μL ddH2O (RNase- and DNase-free).

[0031] Library quality control and quantitative analysis: The cDNA concentration of the library was measured using a quantitative fluorescence analyzer (specifically, Qubit); and the peak shape of the library was quality controlled. The quality control results are as follows: Figure 2 As shown, a trace amount of cellular genomic DNA G-quadruplex cDNA library was successfully constructed and can be used for subsequent sequencing analysis. The sequenced library was compared and analyzed; the distribution and enrichment of G-quadruplex DNA in the 4-cell embryo genome are shown below. Figure 3 As shown.

[0032] In summary, the present invention has the following important practical significance: (1) The distribution and enrichment of genomic DNA G-quadruplexes in trace cell samples can be accurately detected by the G4tacc-seq library preparation method proposed in this invention.

[0033] (2) The G4tacc-seq provided by the present invention can be used to evaluate oocyte quality and early embryonic development potential, and to determine infertility caused by poor oocyte quality and infertility caused by early embryonic development arrest.

[0034] (3) Using the G4tacc-seq library construction method proposed in this invention, drugs for treating fertilized egg development arrest can be screened and prepared at the whole genome level, reducing the occurrence of drug side effects caused by one-sided information analysis.

[0035] (4) Samples of living lesion tissue can be taken and a library can be built using G4tacc-seq to detect the distribution and enrichment of abnormal genomic DNA G-quadruplexes in the lesion tissue, so that the diagnostic results are based on the actual physiological state.

[0036] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a library for detecting G-quadruplex structures in oocytes, early embryos, and trace cells, characterized in that: Includes the following steps: S1. Prepare equipment and consumables, including a stereomicroscope with a heated stage, an oocyte aspiration needle, a pipette, and low-absorption centrifuge tubes; ethanol, ddH2O, AMPure magnetic beads, transposase reaction buffer, PCR amplification enzyme, PCR index primers, digitalis saponins, carrier RNA, and pA / G-Tn5 transposase. S2. Collect samples and count cells. Cells should be freshly harvested, as frozen cells cannot guarantee the good quality of the G4tacc-seq library. Cells should be intact and in a homogeneous single-cell suspension. Oocytes, early embryos, and trace cells to be tested are washed, separated, and counted using an oral pipette and placed on ice for later use. S3, BG4 and pA / G-Tn5 complex were prepared by incubating at 95°C for 5 minutes to fully dissolve 5% digitalis saponin. 1 μL of 5% digitalis saponin was added to 1 mL of buffer 1 (B-1K) to prepare D-buffer 1 (DB-1K) and vortexed to mix. 7 μL of DB-1K, 0.5 μL of pA / G-Tn5 transposase and 0.5 μg of BG4 antibody were added to a 200 μL low-adsorption centrifuge tube and vortexed at 4°C for 30 minutes (400 rpm). S4, prepare the cell lysis buffer. Add 50-100 cells to the wall of a 200μL low-adsorption centrifuge tube using a pipette. To ensure the total volume of liquid and cells is <1μL, use a pipette to aspirate excess liquid. Add 6μL of DB-1K to the cells and incubate on ice for 10 minutes, gently vortexing once every 2.5 minutes. S5, Chromatin cleavage: Add 35 μL of DB-1K to the cell lysis buffer to ensure the stability of the DNA G-quadruplex structure during the chromatin cleavage reaction. Then add BG4 antibody and pA / G-Tn5 complex. Add 12.5 μL of pre-warmed transposase reaction buffer. Vortex at 37°C (400 rpm) in a thermal mixer for 30 minutes to perform the genome chromatin cleavage reaction. Add 2 μL of 10% SDS to the cell lysis buffer after chromatin cleavage and incubate at 55°C for 10 minutes to terminate the chromatin cleavage reaction. S6, Genomic DNA Purification: Allow AMPure magnetic beads to reach room temperature for 20 minutes before use. Add 1 μL of Carrier RNA and 2 μL of internal control DNA (optional) to the termination reaction mixture, then add 150 μL of AMPure magnetic beads. Mix well by pipetting and incubate at room temperature for 5 minutes. Place the low-adsorption centrifuge tube containing the termination reaction product on a magnetic rack and precipitate the magnetic bead-DNA complex for 5 minutes. Keep the low-adsorption centrifuge tube on the magnetic rack, discard the supernatant, and wash the magnetic bead-DNA complex twice with 80% ethanol. After the magnetic bead-DNA complex has air-dried, dissolve the magnetic bead-DNA complex in nuclease-free ddH2O and incubate at room temperature for 3 minutes. Place the dissolved mixture on a magnetic rack to precipitate the magnetic beads for 2 minutes. Collect the supernatant as the target genomic DNA fragment after transposition. S7, PCR amplification reaction: Prepare 50 μL of PCR reaction mixture containing PCR amplification enzyme in a 200 μL PCR tube to amplify the target DNA fragment. S8, the amplified library was purified using AMPure magnetic beads. Large fragments were filtered out using 0.4× magnetic beads, and small fragments were filtered out using 1.7× magnetic beads. The library was then eluted with 20 μL of ddH2O. S9. Library quality control and quantification: The cDNA concentration of the library was measured using a fluorescence quantitative analyzer; and the peak shape of the library was also checked. Sequencing data were compared and analyzed to quantify the enrichment of G-quadruplex DNA structures in trace amounts of early embryonic cell genomes.

2. The method according to claim 1, characterized in that: The components of buffer 1 (B-1K) in S3 include 10 mM Tris hydrochloride at pH 7.4, 150 mM potassium chloride, 0.5 mM spermidine, and a 1X mixture of protease inhibitors that are EDTA-free.

3. The method according to claim 1, characterized in that: The 50 μL PCR reaction mixture in S7 is formulated as follows: 29 μL DNA product, 10 μL 5× polymerization reaction buffer, 5 μL i5 index, 5 μL i7 index, and 1 μL polymerase.

4. The method according to claim 3, characterized in that: The sample in S7 is provided with a barcode Index N5XX corresponding to the i5 index and a barcode Index N7XX corresponding to the i7 index.

5. A method according to claim 1, characterized in that: The reaction conditions for the PCR amplification reaction in S7 are as follows: Step 1: 72℃ for 3 min, 1 cycle; Step 2: 98℃ for 30 sec, 1 cycle; Step 3: 98℃ for 15 sec, 60℃ for 30 sec, 72℃ for 3 min, 16-25 cycles; Step 4: 72℃ for 5 min, 1 cycle; Step 5: Hold at 16℃.

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