Method for constructing library based on large-fragment nucleic acid molecules

Abstract

The invention relates to a method for constructing a library based on large-fragment nucleic acid molecules. The invention also relates to a kit used in the method, and a library obtained by using the method.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for constructing a library based on large fragment nucleic acid molecules. On the basis that both ends of large fragment nucleic acid molecules such as cDNA are bound by cellular oligonucleotide labels, multiple positions within the full length range of the large fragment nucleic acid molecules are also bridged with cellular oligonucleotide labels to form a bridge-like structure, so as to obtain nucleotide fragments of different lengths with oligonucleotide labels for library construction. The present invention also relates to the use of the library in detecting the gene mutation ratio in a genome. Background Art

[0002] Single cell sequencing technology is to detect the gene information of a single cell through next-generation sequencing technology, so as to further understand the state of a single cell in a specific microenvironment. Single-cell RNA sequencing (scRNA-seq) technology is an important member of single cell sequencing. It can sequence and analyze thousands or tens of thousands of cells isolated from solid tissues or body fluids at the RNA level in units of cells, so as to obtain the biological characteristics of these cells in a certain state. ScRNA-seq is particularly suitable for studying tissues or organs with high heterogeneity, such as early embryonic development and tumor cell populations. Spatial transcriptomics technology can obtain the gene expression patterns at the original positions in space in tissues, while recording transcriptional heterogeneity and spatial coordinates.

[0003] Regarding the scRNA-seq technology, since it was first reported by Professor Tang Fuchou from Peking University in 2009, it has been revolutionized and optimized almost every year, making very rapid progress. In 2012, technologies such as SMART-seq, SMART-seq2, and SMART-seq3 jointly developed by American and Swedish scientists (Ramskold, D. et al. Full-length mRNA-seq from single-cell levels of RNA and individual circulating tumor cells. Nat. Biotechnol. 30, 777–782 (2012); Picelli S, Bjorklund AK, Faridani OR, Sagasser S, Winberg G, Sandberg R. SMART-seq2 for sensitive full-length transcriptome profiling in single cells. Nat Methods. 2013;10(11):1096–1098) were able to detect the full length of mRNA and improve the coverage of transcript sequences. However, its drawbacks were also obvious. It was necessary to isolate single cells one by one and conduct separate experiments. This strategy had low throughput, took a long time, and was costly.

[0004] To break through this technical barrier, single-cell identification technology based on barcode labels emerged. Its core technology is to add a unique barcode label to each cell and regard nucleic acid fragments carrying the same barcode label as fragments from the same cell. Cells are labeled by Barcode, and transcripts are labeled by UMI (Unique Molecular Identifier).

[0005] Technologies represented by this strategy include Drop-seq and inDROP (Macosko EZ, Basu A, Satija R, Nemesh J, Shekhar K, Goldman M, et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell. 2015;161:1202–1214; Klein AM, Mazutis L, Akartuna I, Tallapragada N, Veres A, Li V, et al.. Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells. Cell (2015) 161(5):1187–12015), which can complete library construction for a large number of cells at one time. The emergence of these two single-cell sequencing technologies has made it possible to detect thousands of single cells quickly and relatively inexpensively, marking the entry of scRNA-seq into the high-throughput era. However, the disadvantage of Drop-seq is its relatively low cell capture rate and high cost.

[0006] In 2016, the technology of 10X Genomics (Mostovoy Y, Levy-Sakin M, Lam J et al.. A hybrid approach for de novo human genome sequence assembly and phasing. Nat Methods 2016;13:587–90) emerged, and its principle is very similar to that of Drop-seq.

[0007] In 2017, BD Rhapsody released its scRNA-seq technology in a research article on the brain nerves in Nature and commercialized this technology (Birey F, Andersen J, Makinson CD, et al. Assembly of functionally integrated human forebrain spheroids[J]. Nature, 2017, 545(7652):54).

[0008] In 2018, scientists from the Allen Institute in the United States and the University of Washington jointly developed the SPLiT-seq technology (Rosenberg AB, Roco CM, Muscat RA, et al. Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding. Science, 2018, 360(6385):176-182). This technology performs 4 rounds of "shuffling" on a large number of cells (theoretically up to 1 million). In each round of the "shuffling" step, the cells are first divided into different pools, and the mRNA of the cells in each pool is labeled with a unique barcode, and then they are mixed together for the next round of "shuffling". After four rounds of "shuffling", the mRNA of each cell basically contains its own unique barcode combination.

[0009] Single-cell sequencing technology is not only widely used in scientific research, but also has great clinical application prospects. For example, cancer is one of the greatest threats to human health. By performing scRNA-seq on tissue specimens of cancer patients, it is possible to analyze the heterogeneity of tumors, helping patients achieve individualized diagnosis, treatment, monitoring and prognosis assessment.

[0010] Although single-cell sequencing technology has such broad application prospects, it still faces challenges in many aspects, especially the short gene coverage length, which can only analyze the 3' region of genes. 10X Genomics subsequently developed a detection kit for the 5' region, but the ability to cover a wider range of genes has not been improved, not meeting the clinical needs for more gene information.

[0011] Limited by the read length range of next-generation sequencing, current single-cell transcriptome sequencing can only detect the 3' or 5' region of genes (referred to as 3' sequencing or 5' sequencing), and cannot well cover the middle and full length of mRNA. Therefore, it is difficult to analyze the true situation of cells with gene mutations.

[0012] When analyzing the heterogeneity of tumor mutant cells using single-cell sequencing, currently only the differences in expressed genes can be analyzed to classify the cells. Therefore, the obtained expression information is limited to one-sidedly analyzing the heterogeneity of tumors, without involving cell mutations, neither being able to analyze which cells express which mutations, nor being able to analyze whether the expression of other genes in the cells expressing mutations has changed.

[0013] Analyzing the expression profiles of mutant cells helps to analyze the clinical functions, pathogenic mechanisms of genetic disease mutations, detection targets, treatment targets, etc. of diseases caused by gene mutations.

[0014] There are also reports on detecting mutant expression profiles through third-generation sequencing. However, due to the accuracy and high cost of third-generation sequencing, it is currently difficult to be practically applied. In addition, third-generation sequencing has sequencing errors in the cell marker sequences themselves and in the mutations, resulting in a relatively high error background and inaccurate analysis.

[0015] There is still a need in the art to develop new library construction methods that can achieve high-throughput sequencing with a longer gene region coverage length. Summary of the Invention

[0016] The inventors have developed a method for preparing a solid matrix with random nucleotide fragments of a large fragment nucleic acid molecule based on the large fragment nucleic acid molecule and using it for library construction. The core of the method of the present invention lies in, on the basis of labeling both ends of a large fragment nucleic acid molecule such as cDNA, by means of two 3'-ends protruding on the duplex formed by two bridging oligonucleotides, enabling one or more positions in the middle (not at both ends) of the large fragment nucleic acid molecule to form a bridge structure with oligonucleotide labels, so that multiple positions within the full length range of the large fragment nucleic acid molecule can be captured on a solid matrix. Through subsequent amplification, a solid matrix with random nucleotide fragments of the large fragment nucleic acid molecule is obtained, and the set of random nucleotide fragments carried on the same solid matrix can cover the full length range of the large fragment nucleic acid molecule, solving the problem that traditional library construction methods cannot cover the full length well, thus completing the present invention.

[0017] In a first aspect, the present invention relates to a method for preparing a solid matrix with random nucleotide fragments of a large fragment nucleic acid molecule, the random nucleotide fragments being linked to oligonucleotide labels on the solid matrix, the method comprising:

[0018] 1) Using a solid matrix with multiple oligonucleotide labels to capture the large fragment nucleic acid molecule;

[0019] 2) Preparing single-stranded DNA based on the captured large fragment nucleic acid molecule;

[0020] 3) Using a first bridging oligonucleotide and a second bridging oligonucleotide to link one or more positions on the single-stranded DNA to multiple oligonucleotide labels on the solid matrix respectively, the first bridging oligonucleotide and the second oligonucleotide having complementary 5' parts to each other, the remaining 3' part of the first bridging oligonucleotide being reverse complementary to the oligonucleotide label, and the remaining 3' part of the second bridging oligonucleotide being a random sequence and complementary binding to multiple random positions on the single-stranded DNA through the random sequence;

[0021] 4) At the multiple random positions, using the single-stranded DNA as a template and extending along the 3' end of the second bridging oligonucleotide to synthesize multiple random nucleotide fragments, thereby enabling the solid matrix to carry multiple random nucleotide fragments of the large fragment nucleic acid molecules linked to the oligonucleotide label.

[0022] In a second aspect, the present invention relates to a solid matrix carrying random nucleotide fragments of the large fragment nucleic acid molecules prepared by the method of the first aspect.

[0023] In a third aspect, the present invention relates to a method for constructing a sequencing library, comprising:

[0024] Using the random nucleotide fragments of the large fragment nucleic acid molecules carried on the solid matrix of the second aspect as a template for amplification to obtain an amplification product containing the oligonucleotide label and random nucleotide fragments; and

[0025] Adding sequencing adapters to the amplification product.

[0026] In a fourth aspect, the present invention relates to a sequencing library prepared by the method of the third aspect.

[0027] Preferably, the methods of the first and third aspects of the present invention are combined with a library construction method for enriching mutation regions.

[0028] The present invention has at least the following advantages:

[0029] 1) The method of the present invention is applicable to various scenarios and has flexible variations. For example, the method of the present invention can be used to capture different types of large fragment nucleic acid molecules, including mRNA and DNA, as the basis for library construction. Also, for example, the solid matrix of the present invention is not limited to microbeads, and oligonucleotide label clusters can also be formed on the surface of other solid materials to complete the capture, labeling, and subsequent analysis of large fragment nucleic acid molecules.

[0030] 2) The library obtained by the method of the present invention is particularly suitable for the determination of single-cell mutation expression profiles and spatial transcriptomes. Taking single-cell transcriptome sequencing as an example, it is necessary to capture mRNA from a single cell. After capturing the mRNA of a single cell and adding Barcode and UMI to the 3' end, the methodology of the present invention can further add Barcode and UMI to the nucleotide fragments corresponding to the 5' end of the mRNA on the same solid matrix such as microbeads, or can also add Barcode and UMI to the nucleotide fragments corresponding to the middle segment of the mRNA.

[0031] 3) The library constructed by the method of the present invention can cover the full-length information of large fragment nucleic acid molecules such as mRNA, without missing mutations, so as to accurately discover the mutations expressed in each cell, and is suitable for obtaining the expression pattern of gene mutations in a large number of cells (single-cell mutation expression profile). When the method is used for constructing a next-generation sequencing library, since next-generation sequencing is short-read sequencing, the occurrence of sequencing errors can be greatly avoided. The library can be used for single-cell transcriptome or spatial transcriptome sequencing.

[0032] 4) The single-cell transcriptome library obtained by the method of the prior art can only detect the 3' transcriptome. In contrast, the library of the present invention obtains more comprehensive and rich information and can be used to screen libraries for information such as 3' transcriptome and 5' transcriptome. For example, when labeling the 5' part and the middle part of a large fragment nucleic acid molecule with a bridging oligonucleotide, it can be analyzed whether a fixed universal sequence is included in the sequences generated by amplifying different regions, based on sequencing to determine whether a random nucleotide fragment comes from the 5' end or the middle region of mRNA. Similarly, it can also be identified whether this fragment is a 3' region. For example, bridging oligonucleotides can be used to increase different recognition sequences, so as to be able to distinguish the 5' region and the middle region in the amplification product; and / or Tag primers can be used to increase different recognition sequences, so as to be able to distinguish the 5' region and the 3' region in the amplification product. Further, the data of the 3' region can be extracted by virtue of this recognition ability, and the information of the 3' region alone can be used to analyze the transcriptome situation, which is convenient for matching with the existing single-cell sequencing results at the present stage for consistency analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is an example of the method of the present invention, in which the captured large fragment nucleic acid molecule is mRNA. (A) Microbead capture step; (B) Tag primer annealing step; (C) Denaturation washing step; (D) Template bridging oligonucleotide mixture annealing step; (E) Extension step using DNA polymerase; (F) Denaturation washing step; (G) Termination primer annealing step.

[0034] Figure 2 is a schematic diagram of the full-length labeled cDNA library construction method of the present invention. From top to bottom are the diagrams of the steps of amplifying using amplification primers, the steps of fragmenting and semi-linking sequencing adapters, and the steps of enriching the mutant region for library construction.

[0035] Figure 3 Schematically shows an embodiment of capturing a large fragment nucleic acid molecule and preparing single-stranded DNA based on the captured large fragment nucleic acid molecule in the method of the present invention. In this embodiment, the large fragment nucleic acid molecule is captured by oligonucleotide labeling and capture auxiliary oligonucleotide, the single-stranded gap is repaired by a ligase reaction, and then the capture auxiliary oligonucleotide is removed by annealing, and the captured large fragment nucleic acid molecule itself is retained.

[0036] Figure 4 Figure 4 illustrates an embodiment of the method of the present invention for capturing large fragment nucleic acid molecules and preparing single-stranded DNA based on the captured large fragment nucleic acid molecules. In this embodiment, the large fragment nucleic acid molecules are captured by oligonucleotide labeling itself, the complementary strand is obtained by extension, and then the large fragment nucleic acid molecules serving as templates and their extension products are removed by annealing, and the complementary strand linked to the oligonucleotide labeling is retained.

[0037] Figure 5 It is a graph of the gene body coverage rate of the library of the 5.1 expression profiling library construction group, and the X-axis represents the relative position of the overall gene structure.

[0038] Figure 6 It is the sequencing result of the 5.1 expression profiling library construction group library, and the graph shows the total number of genes, the number of RNA molecules, and the mitochondrial ratio of library 5.1 from left to right.

[0039] Figure 7 It is a graph of the cell clustering distribution of the sequencing result of library 6.1.

[0040] Figure 8 It is a graph of the gene expression of the cells measured in library 6.1. The vertical axis shows the gene names, with bright colors indicating high expression and dark colors indicating low expression.

[0041] Figure 9 It is a graph of the mutant cell clustering distribution of the sequencing result of library 7.3.

[0042] Figure 10 It is a graph of the gene expression of the cells measured in library 7.3. The vertical axis shows the gene names, with bright colors indicating high expression and dark colors indicating low expression.

[0043] Figure 11 It shows the flowcharts of the four schemes exemplified in Example 1, Example 2, Example 3, and Example 4 of the present invention. Detailed Description of the Invention

[0045] I. Definitions

[0046] "Large fragment nucleic acid molecule" refers to a nucleic acid molecule with a length of more than 500 bp in the context of the present invention, including DNA and RNA.

[0047] "Random nucleotide fragment" refers to a nucleotide fragment amplified using a part of the "large fragment nucleic acid molecule" as a template in the context of the present invention, and its length is shorter than the "large fragment nucleic acid molecule" of the present invention. "Random" means that it comes from an unspecified position in the "large fragment nucleic acid molecule", rather than meaning that the nucleotide composition of the nucleotide fragment is randomly arranged.

[0048] "Solid matrix" in the context of the present invention refers to a solid material suitable for attaching nucleotides.

[0049] "Oligonucleotide label" in the context of the present invention refers to an oligonucleotide carrying sequence identity recognition information of nucleotides, such as a labeled sequence, a Barcode sequence, which may further include other elements, such as a linker sequence for attaching to a solid matrix, and / or a sequence for attaching to other sequences, such as a poly-T sequence for capturing mRNA.

[0050] "Bridging oligonucleotide" in the context of the present invention specifically refers to an oligonucleotide that bridges a random site on a large fragment nucleic acid molecule and an oligonucleotide label on a solid matrix through reverse complementary relationships.

[0051] II. Overview of Library Construction Method

[0052] The present invention provides a method for preparing a solid matrix with random nucleotide fragments of a large fragment nucleic acid molecule on a solid matrix, wherein the random nucleotide fragments are attached to oligonucleotide labels on the solid matrix, and the method includes:

[0053] 1) Capturing the large fragment nucleic acid molecule using a solid matrix with multiple oligonucleotide labels;

[0054] 2) Preparing single-stranded DNA based on the captured large fragment nucleic acid molecule;

[0055] 3) Using a first bridging oligonucleotide and a second bridging oligonucleotide to attach one or more positions on the single-stranded DNA to multiple oligonucleotide labels on the solid matrix respectively, wherein the 5' parts of the first bridging oligonucleotide and the second oligonucleotide are complementary to each other, the remaining 3' part of the first bridging oligonucleotide is reverse complementary to the oligonucleotide label, and the remaining 3' part of the second bridging oligonucleotide is a random sequence and binds complementarily to multiple random positions on the single-stranded DNA through the random sequence;

[0056] 4) At the multiple random positions, using the single-stranded DNA as a template and extending along the 3' end of the second bridging oligonucleotide to synthesize multiple random nucleotide fragments, thereby enabling the solid matrix to carry multiple random nucleotide fragments of the large fragment nucleic acid molecule attached to the oligonucleotide labels.

[0057] In some embodiments, the large fragment nucleic acid molecule is a DNA molecule or an RNA molecule. The length of the large fragment nucleic acid molecule can be from about 1 kb to about 20 kb. For example, when the large fragment nucleic acid molecule is an RNA molecule such as mRNA, it can have a length of about 1 kb or longer. For example, when the large fragment nucleic acid molecule is a DNA molecule, it can have a longer length than the mRNA molecule, such as from about 1 kb to about 20 kb, preferably from 5 kb to about 20 kb, more preferably from 10 kb to 20 kb, or a longer length.

[0058] The composition of the oligonucleotide label on the solid matrix can be determined according to the specific application of the present invention. Generally speaking, the oligonucleotide label contains a Barcode sequence. In some embodiments, each oligonucleotide label on the same solid phase matrix contains the same Barcode sequence. In some embodiments, the oligonucleotide labels on the same solid phase matrix also contain respective different label sequences. In order to be linked to the solid matrix, the oligonucleotide label also contains a linker sequence.

[0059] In some embodiments, the capture in step 1) is achieved by partial reverse complementarity between the large fragment nucleic acid molecule and the oligonucleotide label. Therefore, the sequence of the free end of the oligonucleotide label needs to be reverse complementary to the end of the large fragment nucleic acid molecule to be captured. This method is particularly suitable for capturing mRNA molecules because they all have polyA sequences at their ends, so the ends of the oligonucleotide labels can be designed as polyT sequences that are reverse complementary to them. In this way, the capture in step 1) is achieved by reverse complementarity between the 3'-end of the large fragment nucleic acid molecule and the 3'-end of the oligonucleotide label. In this embodiment, the large fragment nucleic acid molecule captured can also be DNA.

[0060] In some embodiments, the capture in step 1) is carried out by using a capture-assisting oligonucleotide, and both sides of the capture-assisting oligonucleotide are complementary to the oligonucleotide label and the large fragment nucleic acid molecule, such as Figure 3As shown. In a preferred embodiment, the large fragment nucleic acid molecule to be captured is pre-treated to ligate a sequence complementary to the capture auxiliary oligonucleotide to its 5' end. The method of adding one or more bases to the end of a nucleotide sequence is well known to those skilled in the art. The length of the capture auxiliary oligonucleotide can be 4 - 70 bases, preferably 20 - 40 bases, such as about 30 bases. The length of the part of the capture auxiliary oligonucleotide complementary to the oligonucleotide label can be 4 - 40 bases, preferably 10 - 20 bases, such as about 15 bases. The length of the part of the capture auxiliary oligonucleotide complementary to the large fragment nucleic acid molecule can be 4 - 40 bases, preferably 10 - 20 bases, such as about 15 bases. In a preferred embodiment, the capture auxiliary oligonucleotide itself does not form a dimer or hairpin structure. In a preferred embodiment, the capture auxiliary oligonucleotide only contains the part complementary to the oligonucleotide label and the part complementary to the large fragment nucleic acid molecule, that is, it is composed of these two parts, and the sequences of these two parts do not overlap with each other. In such an embodiment, it is preferred that the captured large fragment nucleic acid molecule is DNA.

[0061] In step 2), the single-stranded DNA is prepared by reverse transcription reaction, ligase reaction or DNA polymerase reaction. In some embodiments, when the captured large fragment nucleic acid molecule is RNA, a complementary cDNA strand is synthesized by reverse transcription reaction as the single-stranded DNA. In some embodiments, when ligating the oligonucleotide label and the large fragment through the capture auxiliary oligonucleotide, since there is a gap between the two nucleic acids to be ligated, the gap is filled by ligase reaction. In some embodiments, after capturing the DNA molecule by reverse complementarity with the oligonucleotide label, the single-stranded DNA is prepared by DNA polymerase reaction.

[0062] Preferably, after the synthesis of the single-stranded DNA is completed, other nucleic acid molecules such as their complementary strands and free nucleic acid molecules are removed by denaturing the double-strand to unwind, etc., except for the single-stranded DNA and the oligonucleotide label ligated thereto.

[0063] In some embodiments, in step 3), the ligation is carried out by DNA polymerase reaction or ligase reaction. In a preferred embodiment, the ligation is carried out by ligase reaction.

[0064] In some embodiments, in step 4), the extension is carried out by DNA polymerase reaction. In some embodiments, the DNA polymerase reaction is a DNA displacement polymerase reaction.

[0065] In some embodiments, after step 4), the double strand is denatured to unwind the strands, and only the strand containing the oligonucleotide label and the random nucleotide fragment attached to the solid matrix is retained.

[0066] In some embodiments, each of the multiple oligonucleotide labels carried by the solid matrix contains a Barcode sequence and a label sequence. When the long fragment nucleic acid molecule to be captured is mRNA, preferably the end of the oligonucleotide label contains a poly-T sequence.

[0067] In some embodiments, before ligation by the first bridging oligonucleotide and the second bridging oligonucleotide in step 3), the 3'-end of the single-stranded DNA end is modified to be reverse complementary to the 3'-end of the oligonucleotide label.

[0068] In some embodiments, before ligation by the first bridging oligonucleotide and the second bridging oligonucleotide in step 3), the oligonucleotide labels on the solid matrix that have not captured the large fragment nucleic acid molecule are modified so that their 3'-ends carry a tag sequence; and the 3'-end of the single-stranded DNA end is modified to be at least partially reverse complementary to the tag sequence; and the tag sequence is at least partially reverse complementary to the remaining 3'-portion of the first bridging oligonucleotide. The tag sequence can be introduced by a tag primer.

[0069] In the method of the present invention, the bridging oligonucleotide and optionally the tag sequence can be used as markers indicating which part of the original long fragment nucleic acid molecule the finally obtained random nucleotide fragment comes from. For example, as Figure 1 shown, when the formed random nucleotide fragment contains a sequence from the bridging oligonucleotide, it indicates that it comes from the middle of the original long fragment nucleic acid molecule; when the formed random nucleotide fragment does not contain a sequence from the bridging oligonucleotide, it indicates that it comes from the 5'-end or 3'-end of the original long fragment nucleic acid molecule. In the case of further using a tag sequence, for example, when a tag sequence is introduced by a tag primer, it can be distinguished whether the formed random nucleotide fragment comes from the 5'-end or 3'-end of the original long fragment nucleic acid molecule by whether the finally formed random nucleotide fragment contains the tag sequence. Taking an mRNA molecule as an example, referring to Figure 1 , the random nucleotide fragment formed based on the 3'-end of the original long fragment nucleic acid molecule does not contain a tag sequence, while the 5'-end of the original long fragment nucleic acid molecule contains a tag sequence. When directly capturing a long fragment by a capture-assisted oligonucleotide and a ligation reaction, the situation is reversed, the random nucleotide fragment formed based on the 5'-end of the original long fragment nucleic acid molecule does not contain a tag sequence, while the 3'-end of the original long fragment nucleic acid molecule contains a tag sequence.

[0070] III. Library Construction Method for Transcriptome Sequencing

[0071] In a preferred embodiment, the method of the present invention is used to construct a library for transcriptome sequencing, for example, to construct a library for single-cell transcriptome sequencing. Through the method of the present invention, a sequencing library that covers the full length of mRNA and has Barcodes can be obtained.

[0072] When constructing the library for transcriptome sequencing, the large fragment nucleic acid molecule is mRNA. Compared with the conventional method, one of the characteristics of using the library construction method of the present invention to construct a single-cell transcriptome sequencing library is that it captures the mRNA of a single cell and reverse transcribes it to add 3'-end Barcodes and UMIs, and realizes the addition of 5'-end Barcodes and UMIs (5'-labeling), and the addition of Barcodes and UMIs in the middle segment (middle labeling).

[0073] In the embodiment of the present invention for constructing a library for single-cell transcriptome sequencing, the method for preparing a solid matrix with random nucleotide fragments of the large fragment nucleic acid molecule includes the following steps:

[0074] 1) Using a solid matrix with multiple oligonucleotide labels to capture the large fragment nucleic acid molecule, and the large fragment nucleic acid molecule is mRNA;

[0075] 2) Preparing single-stranded DNA by reverse transcription based on the captured mRNA;

[0076] 3) Using a first bridging oligonucleotide and a second bridging oligonucleotide to connect one or more positions on the single-stranded DNA to the multiple oligonucleotide labels on the solid matrix respectively. The first bridging oligonucleotide and the second oligonucleotide have complementary 5' parts to each other. The remaining 3' part of the first bridging oligonucleotide is reverse complementary to the oligonucleotide label, and the remaining 3' part of the second bridging oligonucleotide is a random sequence and binds complementarily to multiple random positions on the single-stranded DNA through the random sequence;

[0077] 4) At the multiple random positions, using the single-stranded DNA as a template and extending along the 3' end of the second bridging oligonucleotide to synthesize multiple random nucleotide fragments, thereby making the solid matrix carry multiple random nucleotide fragments of the large fragment nucleic acid molecule connected to the oligonucleotide labels.

[0078] In a more specific embodiment, the method includes the following steps:

[0079] 1) Capture large fragment nucleic acid molecules, which are mRNA, using a solid matrix with multiple oligonucleotide tags. The oligonucleotide tags include a Barcode sequence, a marker sequence, and a poly-T sequence. After contact, among the multiple oligonucleotide tags, some oligonucleotide tags capture mRNA by binding the poly-T sequence to the poly-A of mRNA, and the remaining oligonucleotide tags are free oligonucleotide tags that do not capture mRNA;

[0080] 2) Prepare single-stranded DNA as the first-strand cDNA by reverse transcription based on the captured mRNA, and add a Tag’ sequence (Tag’ sequence) to the free end of the first-strand cDNA using a template-switching oligonucleotide (template-switching guiding sequence). Add a Tag sequence (Tag sequence) to the end of the free oligonucleotide tag using a Tag primer.

[0081] The Tag’ sequence is at least partially reverse complementary to the Tag sequence.

[0082] The Tag primer consists of a first fixed universal sequence and a poly-A sequence.

[0083] At least part of the sequence of the template-switching oligonucleotide is the same as the first fixed universal sequence.

[0084] Step 2) includes annealing the poly-A of the Tag primer to the poly-T sequence of the free oligonucleotide tag, forming a Tag that is reverse complementary to the first fixed universal sequence on the free oligonucleotide by extension, and separating the Tag primer from the free oligonucleotide tag by denaturation to obtain a free oligonucleotide with a Tag.

[0085] Add a Tag’ sequence to the free end of the cDNA using a template-switching oligonucleotide, and the Tag’ sequence is at least partially reverse complementary to the Tag.

[0086] 3) Add a first bridging (Bridge) oligonucleotide consisting of a second fixed universal sequence and a first fixed universal sequence, and a second bridging oligonucleotide with a phosphorylated 5’ end consisting of a third fixed universal sequence and a random guiding sequence. The random guiding sequence consists of any one of deoxyribonucleotides A, T, C, G, and the third fixed universal sequence is reverse complementary to the second fixed universal sequence.

[0087] 4) Anneal to cause the first fixed universal sequence of the first bridging oligonucleotide to anneal to the Tag, and the second fixed universal sequence to anneal to the third fixed universal sequence of the second bridging oligonucleotide, the random guiding sequence of the second bridging oligonucleotide to anneal to a random position of the first-strand cDNA, and the Tag' at the cDNA end to anneal to the Tag, thereby forming multiple bridge-like structures;

[0088] 5) Add ligase to ligate the Tag sequence extended from the oligonucleotide label to the third fixed universal sequence;

[0089] 6) Add DNA polymerase to extend the random guiding sequence using the first-strand cDNA as a template;

[0090] 7) Preferably repeat steps 4) to 6) once;

[0091] 8) Add denaturing buffer for denaturation and remove the supernatant;

[0092] 9) Make the oligonucleotide label unable to undergo subsequent extension;

[0093] 10) Add amplification primers for amplification to obtain a full-length labeled cDNA product;

[0094] 11) Add sequencing adapters to the full-length labeled cDNA product in step 10) to obtain a library.

[0095] After step 11), use the library for sequencing.

[0096] In step 1), the solid matrix can be a microbead, a glass slide, a spatial transcriptomics chip, etc. The contact between the mRNA and the solid matrix can be achieved through a process including cell lysis.

[0097] In step 1), an oligonucleotide (Oligo) label is coupled to the solid matrix. The oligonucleotide label enables each encoded microsphere on each microbead or spatial transcriptomics chip to carry a unique nucleotide label like a fingerprint.

[0098] In single-cell sequencing, by containing one cell and one microbead in a micropore, each microbead only captures nucleotides from one cell, and then the sequencing data from the same cell can be identified from the sequencing results through the unique label carried by the microbead. In a preferred embodiment, each microbead carries approximately 1E7 identical oligonucleotide labels.

[0099] In spatial transcriptomics sequencing, by containing detection regions regarded as spots on the chip, each spot captures nucleotides from 0.2 - 70 cells.

[0100] In a specific embodiment, the oligonucleotide label consists of four parts, namely, from 5' to 3', it includes a linker sequence, a Barcode sequence, a label sequence, and a poly-T sequence.

[0101] The linker sequence connects the Barcode sequence for label identification, etc. to the microbeads. The length of the linker sequence is 4 to 70 bases, preferably 10 to 30 bases.

[0102] The Barcode sequence is the same for a single solid matrix such as a microbead, that is, the Barcode sequences on the same microbeads are the same, and the Barcode sequences on different microbeads are different from each other, thereby ensuring that cDNA amplification and library construction amplification can be carried out through the same forward primer subsequently. The oligonucleotide label can be designed in any way so that the Barcode sequences between the same microbeads are exactly the same, and the Barcode sequences between different microbeads are different from each other. The length of the Barcode sequence can be 10 to 50 bases, preferably 10 to 40 bases, more preferably 11 to 35 bases, and even more preferably 12 to 30 bases. The sequence can be an artificial sequence or a non-human source sequence, and the T7 promoter sequence can be cited.

[0103] The label sequence is different for a single solid matrix such as a microbead, and it endows each oligonucleotide label on a single microbead and the subsequently captured sequence with unique identity recognition information. The length of the label sequence can be 5 to 20 bases, preferably 6 to 15 bases, and more preferably 8 to 10 bases.

[0104] In a preferred embodiment, the types of the oligonucleotide label should be no less than 1E5. In a specific embodiment, the Barcode sequence is 12 random bases and the label sequence is 8 random bases.

[0105] The poly-T sequence in the oligonucleotide label is used to capture the 5'-polyadenylate sequence of mRNA. In a preferred embodiment, the poly-T contains at least 15 thymine (T), such as 15 to 30 T, preferably about 20.

[0106] The microbeads can be coupled with the oligonucleotide label by means known in the art. For example, the hydroxyl group on the nucleotide at the 5' end of the oligonucleotide label is replaced with an amino group, and the surface of the microbeads is modified with a carboxyl group, so as to carry out coupling through the condensation reaction of the carboxyl group and the amino group.

[0107] In step 1), the oligonucleotide label in this part that has not captured mRNA after the contact is called the free oligonucleotide label.

[0108] Capturing mRNA refers to using a cellular oligonucleotide labeled with a poly-T structure to capture the poly-A tail at the 3' end of mRNA.

[0109] The 3'-labeling includes the process of reverse transcribing the captured mRNA to achieve the binding of the cellular label to the 3' end of the mRNA and obtain a complete cDNA sequence complementary to the mRNA.

[0110] In step 2), the first-strand cDNA is a single-stranded DNA that is reverse complementary to the mRNA of the cell.

[0111] Part of the sequence of the first template-switching oligonucleotide is the same as the first fixed universal sequence, or the full-length sequence is the same as the first fixed universal sequence, and is used to extend a Tag that is reverse complementary to the first fixed universal sequence on the free oligonucleotide. In one embodiment, the first template-switching oligonucleotide consists of a poly-A sequence and a first fixed universal sequence.

[0112] The length of the first fixed universal sequence is 4 - 100 bases. The first fixed universal sequence and the Tag are reverse complementary.

[0113] All the fixed universal sequences in the present invention are nucleic acid sequences that do not cover the DNA of animals, and the nucleic acid sequences can cover artificial sequences and the DNA of microorganisms.

[0114] The Tag' sequence is a sequence extended from the free end of the cDNA and is at least partially reverse complementary to the Tag.

[0115] The Tag primer consists of the first fixed universal sequence (sequence X in the examples) and a poly-A sequence. The length of the poly-A sequence is 4 - 100 bases. For example, the sequence composition of the Tag primer is as shown in Table 1 of Example 1.

[0116] As step 2), one embodiment is that the reverse transcription process is carried out simultaneously with the annealing process of the poly-A of the Tag primer.

[0117] As step 2), another embodiment is to anneal first and then carry out the reverse transcription process. Specifically, first add the Tag primer for the annealing process to anneal the poly-A of the Tag primer with the poly-T of the oligonucleotide label, and then carry out the reverse transcription process so that cDNA and Tag are synthesized simultaneously.

[0118] As step 2), another embodiment is step 2)', performing a reverse transcription reaction to form cDNA from the mRNA in the oligonucleotide-labeled -mRNA complex in step 1), and adding polyA to the free end of the formed cDNA. In this embodiment, different from the other two embodiments, since polyA is added to the end of the first-strand cDNA, there is no need to use a Tag primer to add a Tag sequence to the free oligonucleotide end, nor to add a Tag' sequence to the end of the cDNA with a template-switching oligonucleotide.

[0119] The process of adding polyA can be carried out with a second template-switching oligonucleotide capable of forming polyA at the end of the cDNA. The sequence composition of the second template-switching oligonucleotide is, for example, a polyT sequence and three riboguanosines (rGrGrG). The length of the polyT sequence is 4 - 100 bases.

[0120] In some embodiments, step 2) can be omitted, that is, the steps of template switching and adding tags are not carried out.

[0121] In step 3), the first bridging oligonucleotide consists of a second fixed universal sequence and a first fixed universal sequence. The second bridging oligonucleotide is a 5'-end phosphorylated oligonucleotide sequence, consisting of a third fixed universal sequence and a random primer sequence.

[0122] The random primer sequence can be a sequence composed of 3 - 50 random bases, preferably a random hexamer to decamer, more preferably a random decamer. The second bridging oligonucleotide anneals to a certain position in the middle part of the cDNA strand using the random primer sequence.

[0123] In particular, when step 2) is step 2)', the first bridging oligonucleotide used in step 3) consists of a second fixed universal sequence and a polyA part.

[0124] The third fixed universal sequence is an artificially designed sequence, 4 - 100 bases in length, and is reverse complementary to the second fixed universal sequence.

[0125] In step 4), the two bridging oligonucleotides can form a duplex with complementary 5'-ends. The duplex has two protruding 3'-ends, where one protruding 3'-end is a random primer sequence, for example, a random primer sequence composed of 10 random bases, and the other protruding 3'-end is a Tag' sequence. At the same time, the bridging oligonucleotide carrying the random primer sequence has a phosphorylation modification at the 5'-end.

[0126] In step 4), particularly when step 2) is step 2)', the process is to perform annealing such that the poly-A of the first bridging oligonucleotide anneals to poly-T, and the second fixed universal sequence anneals to the third fixed universal sequence of the second bridging oligonucleotide, the random guiding sequence of the second bridging oligonucleotide anneals to a random position of the first-strand cDNA, and the poly-A at the cDNA end anneals to poly-T, thereby forming a plurality of bridge-like structures.

[0127] In step 5), particularly when step 2) is step 2)', the process is to ligate the poly-A extended from the oligonucleotide label to the third fixed universal sequence.

[0128] In step 9), the termination primer consists of a first fixed universal sequence and a fourth fixed universal sequence. It is characterized in that the 5'-end of the termination primer is the fourth fixed universal sequence, and the 3'-end is the Tag' sequence. The fourth fixed universal sequence is a random sequence of 4 - 100 bases. The Tag' sequence is at least partially reverse complementary to the Tag sequence.

[0129] In step 9), when ddNTP is added to the end of Tag by DNA polymerase, the DNA polymerase can no longer extend at the end of Tag, which can prevent the oligonucleotide label on this bead from annealing to the cDNA from another bead and thus distinguish the origin of the cDNA.

[0130] As step 9) for stopping extension, another embodiment is to break and shed the end of the free oligonucleotide label having Tag by adding a combination of enzymes. As the combination of enzymes used here, a combination consisting of: a selective hydrolysis enzyme (for forming a DNA strand with a missing base), an apurinic / apyrimidinic endonuclease (for breaking single-stranded DNA), and an exonuclease for degrading single-stranded DNA can be listed. As such a combination, for example, it is a combination of UNG enzyme, APE1 enzyme, and EXO I enzyme.

[0131] The concentrations of UNG enzyme, APE1 enzyme, and EXO I enzyme can be 0.01 U / μL - 10 U / μL, and the action time can be 5 - 60 minutes.

[0132] In step 10), the GC content of the amplification primer is 30% - 70%, it is required to have no 3'-end dimer and hairpin structure, the annealing temperature is 45 - 70 °C, and the length is 15 - 50 bases.

[0133] In the embodiments of the present invention, any one of the fixed universal sequences is preferably designed not to be the same as or complementary to the large fragment nucleic acid molecule to be captured, and preferably not to be the same as or complementary to any sequence of animal origin.

[0134] In step 11), the following three strategies can be used to establish a library for sequencing.

[0135] The first library construction strategy is expression profiling library construction: all cDNA molecules are randomly put into library construction and sequencing analysis.

[0136] Method A for expression profiling library construction successively includes: taking an appropriate amount of cDNA product, directly fragmenting it, ligating adapters in a semi-ligation manner, and performing library amplification to obtain a sequencing library. Method B for expression profiling library construction can also be used. Method B successively includes: amplifying an appropriate amount of cDNA product, then fragmenting it, and ligating adapters in a semi-ligation manner to obtain a sequencing library. The sensitivity of Method B is higher than that of Method A.

[0137] The operation of expression profiling library construction is simple and a large amount of mRNA information can be obtained. However, due to the limitation of the size of sequencing data, some sequences may be missed. To cover more complete mRNA information, the missed partial sequences may require additional high sequencing costs. Therefore, the expression profiling library construction method is only suitable for expression profiling analysis to analyze whether genes are expressed.

[0138] In order to perform additional capture and enrichment of mutations and transcript variants to be analyzed and obtain rich mutant cell information with a smaller amount of data and sequencing cost, the inventors proposed different second and third library construction strategies according to the different numbers of genes and mutation information captured additionally.

[0139] The second library construction strategy is capture probe library construction: by designing capture probes for mRNA molecules, enrich the genes or partial regions of genes that need attention, such as the region of the site of interest, the 3' region of the gene, the 5' region of the gene, etc. from the full-length labeled cDNA, and then perform sequencing, while supplementing the expression profiling data analyzed by the first library construction.

[0140] Capture probe library construction successively includes designing a panel probe set for the genes of interest, first performing capture enrichment on the cDNA product, then amplifying, and fragmenting the amplification product to obtain a sequencing library.

[0141] The advantage of capture probe library construction is that it can analyze the genes of interest at a relatively low cost. The method of using capture probes is generally used when there are more genes of interest. Therefore, the sensitivity of capture probe library construction is higher than that of expression profiling library construction but lower than that of multiplex amplification library construction.

[0142] The third library construction strategy is multiplex amplification library construction, which designs multiplex primers for one or several genes or mutation sites to amplify and enrich the information of key regions.

[0143] Multiple amplification library construction successively includes designing multiple primers for genes or sites of interest, such as regions of sites of interest, CDR3 regions of the immune group, etc. First, amplify and enrich the cDNA products. During amplification and enrichment, semi-nested primers can also be used for multiple rounds of enrichment. Directly amplify and add sequencing adapters to the enriched amplification products to obtain a sequencing library.

[0144] The detection range of multiple amplification library construction is smaller than that of capture probe library construction, but it has the highest sensitivity. In practical applications, the sensitivity of capture probe library construction is about 5 to 10 times that of the first type, while multiple amplification library construction is about 100 to 150 times that of expression profile library construction.

[0145] Specifically, the method for constructing a sequencing library of the present invention may include the following steps:

[0146] 11) Randomly fragment the full-length labeled cDNA products obtained in the above step 10) to obtain fragmented products;

[0147] 12) Add a 3' sequencing adapter to the fragmented products obtained in step 11) to obtain ligation products.

[0148] After step 12), a library for single-cell sequencing is obtained, and the nucleic acid molecules of the ligation products contained therein have adapters suitable for the sequencing platform at both ends.

[0149] In some embodiments, based on the requirements of the sequencing platform, circular products need to be provided as sequencing objects. Therefore, in some embodiments, the library construction method further includes:

[0150] 13) Circularize the ligation products obtained in step 12).

[0151] In step 11), fragment the nucleic acid products obtained in the library construction amplification step. For example, fragmentation can be carried out by mechanical methods or enzymatic treatment, such as by sonication. Preferably, the size of the double-stranded DNA fragments retaining the 5' sequencing adapter after fragmentation is 300 - 500 bp.

[0152] In step 12), add a 3' sequencing adapter to the randomly fragmented products, i.e., "semi-ligation". The 3' sequencing adapter is a double-stranded adapter. In a preferred embodiment, the 3' sequencing adapter ligated by the semi-ligation method is a nucleotide sequence phosphorylated at the 5' end.

[0153] The term "semi-ligation" in the context of the present invention refers to the process of ligating an adapter to a double-stranded DNA, where the two 5' ends of the double-stranded DNA remain unchanged, and the adapter is ligated to the two 3' ends of the double-stranded DNA. In order to ligate the adapter to the 3' end of the amplification product in the correct orientation, the following two semi-ligation methods can be used.

[0154] As the solution for library construction by the semi-joining method, the solution for library construction by the semi-joining method described in the applicant's prior application can be used (see CN111575357A), or a method that directly uses MGI adapters on the basis of this method and realizes adapter switching on the Illumina platform can be used. One of the characteristics of the latter method is that the process of randomly fragmenting cDNA occurs after cDNA amplification but before adding sequencing adapters.

[0155] In a specific embodiment, the 5' end of the 3' sequencing adapter added in step 12) carries a phosphate group. The two strands anneal to form a double-stranded adapter through the reverse complementary part shown by the following underlines. In the formed double-stranded adapter, there is a 3' T sticky end from the antisense strand and a 3' sticky end from the sense strand to prevent the adapter from contacting in the reverse direction and breaking DNA. In the case of not adding A to double-stranded DNA, an adapter without the T sticky end is used, for example, removing the T at the 3' end of the nucleotide sequence.

[0156] After connecting the 3' sequencing adapter, the constructed library is obtained. The obtained library contains double-stranded nucleic acid molecules with sequencing adapters.

[0157] For a sequencing platform applicable to circular nucleic acid samples, after step 12), a cyclization step is further performed on the ligation product, which is step 13) of the present invention. In step 13), the ligation product ligated with two adapters obtained in step 12) is cyclized through the adapter, thereby obtaining the constructed library. The cyclization process can include using a ligase to ligate and using a digestive enzyme to digest. The obtained library contains circular single-stranded nucleic acid molecules.

[0158] The selection of the adapter sequence depends on the sequencing platform to be used.

[0159] In some embodiments, the prepared library or intermediate products during the preparation process can be purified. For example, the products of one or more of the following steps can be purified: the amplification product of the library construction amplification step in step 10), the ligation product of the ligation step in step 12), and the cyclization product of the cyclization step in step 13). The purification process can remove unwanted impurities other than the target product, including nucleic acid fragments, such as small nucleic acid fragments like primer dimers, etc. Conventional purification means in the art can be used. For example, magnetic beads can be used for purification.

[0160] IV. 5’ Labeling and Middle Labeling

[0161] The characteristic of the present invention is to provide a method for realizing 5'-labeling and intermediate labeling of mRNA information in cDNA library construction.

[0162] As a method for 5'-labeling, methods such as binding the sequence information at the 5'-end of mRNA to the oligonucleotide label on the surface of beads through a special linker can be cited. Method A) is illustrated in Examples 1-4: During reverse transcription, a poly-A tail or other similar complementary sequences are added to the cDNA end using template-switching technology, enabling the first-strand cDNA of reverse transcription to bind to the oligonucleotide on the bead surface. However, it is not limited to this, and Method B) can also be used: The oligonucleotide label is ligated to the first-strand cDNA by adding a ligation linker.

[0163] As a method for middle-labeling, a method of indirectly binding the sequence information of the middle segment of mRNA in cDNA to the cell oligonucleotide label through the first-strand cDNA of reverse transcription, a special linker, and random primers (containing random guiding sequences) can be cited.

[0164] The method for middle-labeling can be Method A): Reverse transcription is performed, and then a random primer (Tag primer) with a Tag sequence (the first fixed universal sequence) is annealed to the first-strand cDNA of reverse transcription for extension; meanwhile, another sequence is annealed to the free oligonucleotide label on the bead surface to form a sticky end at the end of the free oligonucleotide label, and the sticky end can recognize the Tag sequence carried by the random primer, thereby ligating the free oligonucleotide label on the surface to the middle region of the cDNA.

[0165] The method for middle-labeling can also be Method B): Random bases are added to the end of the oligonucleotide label on the bead that anneals to the first fixed universal sequence to form an oligonucleotide label with a random sequence at the end, and then the cDNA is bound and extended with the oligonucleotide label with a random sequence at the end to achieve the labeling of the sequence in the middle region of the cDNA.

[0166] In one embodiment, when performing 5'-labeling and middle-labeling, by adding different recognition sequences, sequencing can be used to analyze whether this fragment is the 5'-end or the middle region of mRNA. Similarly, it is also possible to identify whether this fragment is the 3'-end. Further, 3'-end data can be extracted and the 3'-end information can be used alone to analyze the transcriptome situation.

[0167] The methods for 5'-labeling and middle-labeling of the present invention can also be used for DNA fragments, but are preferably used for mRNA. This is because the molecular properties of mRNA are unstable, prone to degradation and fragmentation. When directly performing reverse transcription using random primers, the capture of non-mRNA information (such as rRNA) will increase; moreover, events such as the fragmentation of RNA molecules will make the captured information incomplete.

[0168] The method for 5'-labeling and middle-labeling of the present invention utilizes cDNA for adding 5' and 3' labels, which can completely avoid the above-mentioned defects. Preferably, in the extension step after using primers with random sequences, a high-fidelity strand displacement enzyme is used for extension, which can increase the labeling efficiency.

[0169] As the order of performing 3'-labeling, 5'-labeling, and middle-labeling, one embodiment is as follows: 1) Use beads with cell labels, 2) Capture the mRNA of a single cell, and 3) Perform reverse transcription to add a 3'-label. 4) Then, further perform 5'-labeling and middle-labeling on the same beads. 5) Use the cDNA with completed labeling to construct next-generation sequencing and perform sequencing.

[0170] Another method is: 1) Use beads with cell labels, 2) Capture the mRNA of a single cell, and 3) Perform reverse transcription to add a 3'-label. 4) Then, further perform 5'-labeling and middle-labeling on the same beads. 5) Use the cDNA with completed labeling to enrich mutant genes, construct next-generation sequencing, and perform sequencing.

[0171] Another method is: 1) Use beads with cell labels, 2) Capture the mRNA of a single cell, and 3) Perform reverse transcription to add a 3'-label. 4) Then, further perform 5'-labeling and middle-labeling on the same beads. 5) Use the cDNA with completed labeling to enrich mutant genes using a probe set or using multiple primers, construct next-generation sequencing, and perform sequencing.

[0172] Another method is: 1) Use beads with cell labels, 2) Capture the mRNA of a single cell, and 3) Perform reverse transcription to add a 3'-label. 4) Then, first perform 5'-labeling and then perform middle-labeling. 5) Use the cDNA with completed labeling to enrich mutant genes using a probe set or using multiple primers, construct next-generation sequencing, and perform sequencing.

[0173] Another method is: 1) Use beads with cell labels, 2) Capture the mRNA of a single cell, and 3) Perform reverse transcription to add a 3'-label. 4) Then, perform 5'-labeling by adding adaptor ligation and perform middle-labeling by ligating random primers and extending on the same beads. 5) Use the cDNA with completed labeling to enrich mutant genes using a probe set or using multiple primers, construct next-generation sequencing, and perform sequencing.

[0174] Another method is as follows: 1) Use beads with cell markers; 2) Capture the mRNA of single cells; 3) Perform reverse transcription to add 3'-markers; 4) Then, modify the oligonucleotides on the beads to perform 5'-marking and middle marking; 5) Use the completely marked cDNA, enrich the mutant genes using a probe set or use multiple primers to enrich the mutant genes, construct next-generation sequencing, and perform sequencing.

[0175] Another method is as follows: 1) Use beads with cell markers; 2) Capture the mRNA of single cells; 3) Perform reverse transcription to add 3'-markers; 4) Then, modify the oligonucleotides on the beads, perform 5'-marking by ligating adapters and perform middle marking by ligating random primer extension; 5) Use the completely marked cDNA, enrich the mutant genes using a probe set or use multiple primers to enrich the mutant genes, construct next-generation sequencing, and perform sequencing.

[0176] Another method is as follows: 1) Use beads with cell markers; 2) Capture the mRNA of single cells; 3) Perform reverse transcription to add 3'-markers; 4) Then, modify the oligonucleotides on the beads, perform 5'-marking by ligating adapters and perform middle marking by ligating random primer extension; 5) After repeating step 4 for n times (n >= 2), use the completely marked cDNA, enrich the mutant genes using a probe set or use multiple primers to enrich the mutant genes, construct next-generation sequencing, and perform sequencing.

[0177] V. Method for Judging Whether Mutant Genes are Lost due to Non-expression or Failure to be Enriched Successfully

[0178] Gene expression is not continuous, and there are differences in the expression levels. Therefore, it is necessary to judge whether the mutant gene is not expressed or lost due to unsuccessful enrichment when analyzing data. The inventors found that for accuracy, housekeeping genes can be used as a reference to judge the off-target situation of the captured enrichment or multiple enrichment of the target gene.

[0179] The present invention provides a method for judging whether a mutant gene of interest in the sequencing result is not expressed or lost due to unsuccessful enrichment. The method includes using housekeeping genes such as ACTB, GAPDH, etc. as reference benchmarks to analyze the detection efficiency of expressed genes.

[0180] The method of the present invention uses dual statistics of overall expression analysis and single-cell expression analysis, and can more strictly identify genes with mutant non-expression or know the probability of non-expression of cells. Detailed implementation mode

[0181] To more comprehensively understand and apply the present invention, the present invention will be described in detail below with reference to embodiments and the accompanying drawings. The embodiments are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. The scope of the present invention is specifically defined by the appended claims.

[0182] Embodiment

[0183] Embodiment 1. Full-length labeled cDNA method 1

[0184] In this embodiment, three cell lines, namely Hela, A549 and 293T cells, were mixed in a ratio (3:2:1) as cell materials. mRNA was captured with beads and reverse transcribed into cDNA, and then full-length labeling was performed. The specific protocol is as Figure 1 and 11 shown. The general formula and specific sequences of the primers and probes used are shown in Table 1 below.

[0185] Table 1. General formula and specific sequence information

[0186]

[0187] In the general formula of Table 1 above, sequence X, sequence Y, sequence Z, and sequence W are the first fixed universal sequence, the second fixed universal sequence, the third fixed universal sequence, and the fourth universal fixed sequence respectively, where Y and Z are reverse complementary sequences. In the specific nucleotide sequence, A represents deoxyadenosine base, and both N and J represent any one of A, T, C, and G deoxyribonucleotides (the introduction of J is only for facilitating the distinction between the Barcode sequence and the labeling sequence in oligonucleotide labeling. All Barcode sequences on the same bead are the same, Barcode sequences on different beads are different from each other, and the labeling sequences on the same bead are different from each other; in the sequence listing, it is still represented by N that conforms to the ST.26 standard). P- represents phosphorylation at the 5' end. -NH 2 represents amination at the 3' end.

[0188] Experimental Procedures

[0189] 1. Contact the cells mixed in proportion with the liquid containing beads, capture the cell mRNA, and perform reverse transcription.

[0190] Hela, 293T, and A549 cells were resuspended and counted using PBS. Take the cell mixture and resuspend it in 500 μL of PBS to obtain 3×10 5 cells / mL cell suspension. Add PBS to this cell suspension for further dilution to prepare a cell density of 6.4×10 4 cells / mL and a volume of 500 μL for loading. With a concentration of 1.5×10 7The bead suspension at [[number of beads]] beads / mL was mixed in the single-cell sequencing chip and collected into a collection tube.

[0191] Specifically, lysis buffer (0.5% SDS, 2M NaCl, 10% ficoll) was added to the system containing beads and cells in the collection tube. After lysis, the solution in the collection tube was centrifuged, the supernatant was discarded, the beads in the solution were collected, and the beads were rinsed with 200 μL of reverse transcription buffer (250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl 2 ) and centrifuged again to discard the supernatant. The beads were resuspended using the 50 μL reverse transcription reaction system shown in the following table and incubated at 42 °C for 60 minutes for reverse transcription to obtain cDNA;

[0192] Then, Tag primers were added to the system containing cDNA and annealed. The beads were resuspended using the following Tag primer system and incubated at room temperature with a rotary mixer for 15 min. After completion, the beads were collected by centrifugation and the supernatant was discarded.

[0193] Table 2. Reverse transcription reaction system (50 μL)

[0194]

[0195] Table 3. Tag primer system

[0196] Reagent Name Source Volume Used 20xSSC Buffer NEB 10ul Tag Primer Sangon 5ul NF Water Thermofisher 35ul Total Volume 50uL

[0197] 2. Use DNA polymerase for extension to extend the complementary strand of the oligonucleotide label of the uncaptured mRNA on the beads beyond the first fixed universal sequence.

[0198] The beads were resuspended using the following DNA polymerase extension system and incubated at 37 °C for 30 min.

[0199] Table 4. DNA polymerase extension system

[0200] Reagent Name Source Volume Used 10x NEB Buffer NEB 2ul dNTP mixture Sangon 5ul Klenow NEB 2ul NF Water Thermofisher 11ul Total Volume 20ul

[0201] 3. Add denaturing buffer (NextSeq Rapid DNA Library Preparation Kit, illumina) to wash the beads and remove the supernatant.

[0202] 4. Add the bridge oligonucleotide mixture (the first bridge oligonucleotide, the second bridge oligonucleotide) to the system for annealing.

[0203] Specifically, add the bridging oligonucleotide 1 composed of the second fixed universal sequence and the first fixed universal sequence, and the second bridging oligonucleotide composed of the third fixed universal sequence and the random guiding sequence with a phosphorylated 5'-end, and anneal the first bridging oligonucleotide, the second bridging oligonucleotide with the complementary strand of the first fixed universal sequence extended from the oligonucleotide label or with the first-strand cDNA. After the annealing is completed, multiple bridge-like structures of the oligonucleotide on the bead surface and the cDNA will be formed.

[0204] Resuspend the beads using the following bridging oligonucleotide system and incubate at room temperature with a rotary mixer for 15 min. After completion, centrifuge to collect the beads and discard the supernatant.

[0205] Table 5. Bridging oligonucleotide annealing system

[0206]

[0207] 5. Add the ligase system to ligate the complementary strand (i.e., the tag sequence) of the first fixed universal sequence extended from the oligonucleotide label on the bead surface with the third fixed universal sequence. Resuspend the beads using the following ligase system and incubate at room temperature with a rotary mixer for 30 min. After completion, centrifuge to collect the beads and discard the supernatant.

[0208] Table 6. Ligase system

[0209]

[0210]

[0211] 6. Use DNA polymerase to continue extending the random guiding sequence using the first-strand cDNA as a template. The DNA polymerase extension system in step 6 is the same as that in step 2.

[0212] 7. Repeat steps 3 - 6 once.

[0213] 8. Add the denaturing buffer (the same as above), wash the beads and discard the supernatant.

[0214] 9. Add the termination primer, resuspend the beads using the following termination primer annealing system, and incubate at room temperature with a rotary mixer for 15 min. After completion, centrifuge to collect the beads and discard the supernatant.

[0215] Table 7. Termination primer annealing system

[0216] Reagent Name Source Volume Used 20xSSC Buffer 10ul Termination Primer Sangon 5ul NF Water Thermofisher 35ul Total Volume 50uL

[0217] 10. Extension is carried out using DNA polymerase and ddNTP (dideoxynucleotide) to add ddNTP to the free oligonucleotide ends on the surface of the beads, preventing subsequent extension. Resuspend the beads with the following ddNTP extension system and incubate at 37 °C for 30 min.

[0218] Table 8 ddNTP extension system

[0219] Reagent Name Source Volume Used 10x NEB Buffer NEB 2ul ddNTP mixture Sangon 5ul Klenow NEB 2ul NF Water Thermofisher 11ul Total Volume 20ul

[0220] 11. Add amplification primers and perform amplification using the universal sequence on the beads to obtain full-length labeled cDNA products of different lengths.

[0221] Table 9. Specific sequences of amplification primers

[0222] Name Sequence SEQ ID No. Amplification Primer TAATACGACTCACTATA 8

[0223] Example 2. Full-length labeled cDNA method 2

[0224] In this example, another method is used to obtain full-length labeled cDNA products. In this example, a mixture of three cell lines, Hela, A549, and 293T cells (3:2:1), is also used. Different from Example 1, after capturing mRNA in step 1, a tag primer is first added and annealed, then reverse transcription is carried out, and then a denaturing buffer is added. After that, all other steps are the same as those from step 4 and later in Example 1. The general step flow is as Figure 11 shown.

[0225] Experimental Procedures

[0226] 1. The same as step 1 in Example 1, except that a tag primer is first added for annealing to anneal the tag primer with the oligonucleotide label, and then reverse transcription is carried out to simultaneously synthesize cDNA and the complementary strand of the tag primer.

[0227] Resuspend the beads with the following system and incubate at room temperature using a rotary mixer for 15 min. After completion, centrifuge to collect the beads and discard the supernatant.

[0228] Table 10. Tag primer system

[0229] Reagent Name Source Volume Used 20xSSC Buffer NEB 10ul Tag Primer Sangon 5ul RNase inhibitor Tarkara 1ul NF Water Thermofisher 34ul Total Volume 50uL

[0230] 2. The same as step 3 in Example 1.

[0231] 3 - 10. The same as steps 4 - 11 in Example 1 to obtain full-length labeled cDNA.

[0232] Example 3. Full-length labeled cDNA method 3

[0233] This example uses another method to obtain full-length labeled cDNA products. This example also uses a (3:2:1) mixture of three cell lines, Hela, A549, and 293T cells. Different from Example 1, after the beads capture mRNA in Step 1, reverse transcription is performed, and polyA is added to the end of the first-strand cDNA by template switching. The general step flow is as Figure 11 shown.

[0234] Experimental Procedures

[0235] 1. The same as Step 1 of Example 1, except that after the beads capture mRNA, reverse transcription is performed, and polyA is added to the end of the first-strand cDNA by template switching so that the first-strand cDNA has a polyA tail that can anneal to the polyT labeled on the cell oligonucleotide.

[0236] Specifically, add lysis buffer to the system containing beads and cells in the collection tube. After lysis, centrifuge the solution in the collection tube, discard the supernatant, collect the beads in the solution, and wash and centrifuge again to discard the supernatant with 200 μL of reverse transcription buffer (250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2). Resuspend the beads with the 50 μL reverse transcription reaction system shown in the following table and incubate at 42 °C for 60 minutes for reverse transcription to obtain cDNA;

[0237] Table 11. Reverse transcription reaction system

[0238]

[0239] After obtaining cDNA, add denaturation buffer, centrifuge to collect the beads, and discard the supernatant. Resuspend the beads with 4xSSC buffer and incubate at room temperature with a rotary mixer for 15 min.

[0240] 2 - 11. The same as Steps 2 - 11 of Example 1 to obtain full-length labeled cDNA.

[0241] Example 4. Full-length labeled cDNA method 4 (enzyme treatment)

[0242] This example also uses a (3:2:1) mixture of three cell lines, Hela, A549, and 293T cells. Different from Example 1, in this example, different bridging oligonucleotides are used, and instead of the termination primer and ddNTP, an enzyme treatment step is used. The primers used are shown in Table 12 below.

[0243] Table 12. Sequence information

[0244]

[0245] In this embodiment, sequence X is the sequence X in bridging oligonucleotide 1; the composition of sequence Y' can refer to the composition of the second fixed universal sequence, and preferably contains multiple discontinuous As; the composition of sequence Z' can refer to the composition of the third fixed universal sequence. Sequence Z' is reverse complementary to sequence Y', and therefore preferably contains multiple discontinuous 2'-deoxyuridine (dU) in the sequence, that is, dU in the sequence. Subsequently, when treated with UNG enzyme, the sequence Z' introduced by bridging oligonucleotide 4 will break at the position of dU, so that this segment of the sequence is digested.

[0246] Experimental Procedures

[0247] 1-3. The same as steps 1-3 of Example 1.

[0248] 4. Add the bridging oligonucleotide mixture (the third bridging oligonucleotide, the fourth bridging oligonucleotide) to the system and perform annealing.

[0249] 5-7. The same as steps 5-7 of Example 1.

[0250] 8. Treat with UNG enzyme, APE1 enzyme and EXO I enzyme. Resuspend the beads with the following system and incubate at 37°C with a rotary mixer for 15 min.

[0251] Table 13. Reaction system

[0252] Reagent Name Source Volume Used 10xCutsmart Buffer NEB 5ul UNG Enzyme NEB 1ul APE1 Enzyme NEB 1ul EXO I Enzyme NEB 1ul NF Water Thermofisher 42ul Total Volume 50uL

[0253] 9. The same as step 11 of Example 1. After completion, centrifuge to collect the beads and discard the supernatant. Obtain the full-length labeled cDNA product.

[0254] Compared with the method of Example 1, the advantage of the enzyme treatment method of Example 4 is that it can greatly save the time used for library construction, from 1 hour to 0.25 hour.

[0255] Example 5. Comparison of different library construction methods

[0256] Using the full-length labeled cDNA product obtained in Example 4 for library construction, the effects of three different ways of library construction were compared.

[0257] 5.1 Expression profile library construction group

[0258] Take an appropriate amount of the cDNA product obtained in Example 4, directly break it, semi-ligate the adapter, and perform library amplification to obtain the sequencing library.

[0259] The steps of breaking and semi-ligating refer to the library construction part in Chinese Patent Application Publication No. CN111575357A to obtain the library of the expression profile library construction group.

[0260] 5.2 Capture Probe Library Construction Group

[0261] Design a panel probe set for genes PLEC, ZCCHC4, and TP53 of interest in T-cell tumors. First, capture and enrich the cDNA products, then amplify them. The amplified products are fragmented and used for library construction.

[0262] The steps of capture and enrichment refer to exome sequencing, such as the IDT xGen Exome Capture Kit. TM ExomeHyb Panel v2, (https: / / sfvideo.blob.core.windows.net / sitefinity / docs / default-source / protocol / xgen - hybridization - capture - of - dna - libraries - protocol.pdf?sfvrsn = 9da6e007_18), page 12 - 18. The obtained products are fragmented and semi - ligated for library construction, i.e., capture probe library construction.

[0263] Use a panel probe set of 980 genes for capture enrichment library construction to obtain a probe library construction group library.

[0264] 5.3 Multiplex Amplification Library Construction Group

[0265] Design multiplex primers for genes PLEC, ZCCHC4, TP53 or loci of interest in T - cell tumors. First, amplify and enrich the cDNA products. During amplification and enrichment, semi - nested primers are used for multiple rounds of enrichment. Directly amplify and add sequencing adapters to the enriched amplified products to obtain a sequencing library. As Figure 4 shown.

[0266] The steps of amplification and enrichment follow AMPLIseq targeted sequencing, such as the IonAmpliSeq Comprehensive Cancer Panel of Thermo Fisher. TM (https: / / www.thermofisher.cn / document - connect / document - connect.html?url = https: / / assets.thermofisher.cn / TFS - Assets%2FLSG%2Fmanuals%2FMAN0006735_AmpliSeq_DNA_RNA_LibPrep_UG.pdf), Page 24 - 31. Perform the operations to obtain a multiplex amplification library construction group library.

[0267] The sequencing results of the expression profiling library construction group of 5.1 are shown in Table 14. Figures 5 - 6 .

[0268] Table 14. Sequencing results

[0269] Data Volume (Gbase) 32.4G Total Number of Genes 22595 Total Number of Cells 5476 Average Number of Genes per Cell 1398 Average Number of UMIs per Cell 3052

[0270] Figure 5 It shows that after using the full-length cDNA labeling technology of the present invention and combining with the expression profiling library construction method of direct fragmentation, 90% of the full-length range of genes can be covered on the expressed genes.

[0271] Example 6. Somatic mutation T cell tumors

[0272] T cell tumors (tumor lymph node tissues of T cell lymphoma patients) were used as cells. Information such as the mutated genes, mutation sites, and mutation frequencies of known cell tumor samples is listed in Table 15.

[0273] Table 15. Mutated genes, mutation sites, and mutation frequencies of known T cell tumors

[0274]

[0275]

[0276] Experimental Procedures

[0277] Using the above samples, single-cell dissociation was performed using the Miltenyi tumor tissue cell dissociation kit, full-length cDNA was labeled in the same manner as in Step 1 of Example 1, and libraries were constructed in the same manner as Steps 5.1, 5.2, and 5.3 in Example 5, respectively, to obtain Library 6.1 (direct fragmentation library), Library 6.2 (library constructed by capturing and enriching with a panel probe set of 980 genes), and 6.3 (multiple semi-nested amplification library of 3 genes). Among them, the primer design sites of Library 6.3 are derived from known mutation sites.

[0278] The three libraries of 6.1, 6.2, and 6.3 were sequenced, and the sensitivity differences of detecting the three mutations in Table 14 were compared to study the sensitivity of different enrichment methods to cell mutations. The sequencing results are shown in Figures 9 - 10 , Table 16.

[0279] Table 16 Results of library construction by multiple semi-nested primer amplification and enrichment

[0280]

[0281] When sequencing the same amount of data, the number of TP53 mutant cells detected during direct fragmentation and library construction (Library 6.1) was 3. When using the panel probe set of 980 genes for capture enrichment and library construction (Library 6.2), the number of TP53 mutant cells detected was 22. When using multiplex semi-nested primer amplification for enrichment and library construction (Library 6.3), the number of TP53 mutant cells detected was 160.

[0282] Table 17. Detection of TP53 Mutations with Different Library Construction Schemes

[0283]

[0284] The results confirmed that the detection sensitivity was the highest when using the multiplex primer amplification method, which was nearly 50 times that of direct fragmentation and library construction. When using the library constructed by this method, it could provide more accurate results when selecting a better cancer immunotherapy plan for patients.

[0285] When comparing the three library construction methods, at the same amount of sequencing data, the detection sensitivity of the data covering more genes was worse. Similarly, for the scheme covering more genes, it was necessary to increase the amount of sequencing data to increase the number of mutant cells detected.

[0286] Example 7. Comparison of Detection Probabilities Using Reference Genes

[0287] In this example, the applicant mixed three types of cells, namely Hela, A549, and 293T cells, in a ratio of 3:2:1, obtained full-length labeled cDNA through the same steps as in Example 1, and constructed a library according to the scheme in 5.3 of Example 5 to obtain Library 7.3.

[0288] Among the Hela, A549, and 293T cells used, the average relative expression level of GAPDH was 10, that of ACTB was 5, and that of SUN1 was 1. Based on all the measured cells, the coverage of GAPDH (NM_002046.7 c.463A) and ACTB (NM_001101.5 c.565C) at the middle position of mRNA was analyzed to judge the probability of detecting cells containing mutations (Cell detection rate = Number of detected cells / Total number of cells × 100%), and the results are shown in Table 18 and Table 19.

[0289] Table 18. Detection of Gene Expression

[0290]

[0291] The results show that, as can be seen from Table 18, the two reference genes were detected in more than 93% of the cells. It can be known that when the method for labeling full-length cDNA of the present invention is combined with the method of constructing a library by multiplex primer amplification, for genes with medium to high expression levels, the cell detection rate is above 93%; for low-expression genes such as SUN1, the cell detection rate is about 30%. This experiment shows that in this method, housekeeping genes can be used as reference genes to correctly evaluate the enrichment status.

[0292] Average relative expression level = sum of UMI counts of total ACTB genes in all cells / total number of cells

[0293] The results show that, as shown in Table 19, in the Hela, A549, and 293T cells used, the average relative expression level of GAPDH was 10, that of ACTB was 5, and that of SUN1 was 1. Analyze the mutation conditions of GAPDH (NM_002046.7 c.463A), ACTB (NM_001101.5 c.565C), and SUN1 (NM_001171945 c.290G) at the middle position of mRNA based on all the measured cells. Among them, it is known that there are no mutations in GAPDH and ACTB, and the SUN1 gene mutates to c.G290C.

[0294] Table 19. Detection of cells with mutated gene expressions

[0295]

[0296] As shown in Table 7, no mutations were found in the ATCB and GAPDH genes in the three cell lines; no mutations were found in the SUN1 gene in the Hela and A549 cell lines either. In the 293T cell line, 384 cells expressed the SUN1 gene, among which 86 cells expressed mutations, and the proportion of mutant cells was 22.4%.

[0297] The sequence numbers and corresponding sequences of the present invention are shown in the following table.

[0298]

Claims

1. A method for preparing a solid matrix with random nucleotide fragments of a large fragment nucleic acid molecule, where the random nucleotide fragments are linked to oligonucleotide tags on the solid matrix, the method comprises: 1) Capturing the large fragment nucleic acid molecule using a solid matrix with multiple oligonucleotide tags; 2) Preparing single-stranded DNA based on the captured large fragment nucleic acid molecule; 3) Using a first bridging oligonucleotide and a second bridging oligonucleotide to link one or more positions on the single-stranded DNA to multiple oligonucleotide tags on the solid matrix respectively. The first bridging oligonucleotide and the second oligonucleotide have complementary 5' parts to each other. The remaining 3' part of the first bridging oligonucleotide is reverse complementary to the oligonucleotide tag, and the remaining 3' part of the second bridging oligonucleotide is a random sequence and binds complementarily to multiple random positions on the single-stranded DNA through the random sequence; 4) At the multiple random positions, using the single-stranded DNA as a template and extending along the 3' end of the second bridging oligonucleotide to synthesize multiple random nucleotide fragments, thereby making the solid matrix carry multiple random nucleotide fragments of the large fragment nucleic acid molecule linked to the oligonucleotide tags.

2. The method according to claim 1, wherein the large fragment nucleic acid molecule is a DNA molecule or an RNA molecule.

3. The method according to claim 1 or 2, wherein the length of the large fragment nucleic acid molecule is about 1 kb to about 20 kb.

4. The method according to any one of claims 1-3, wherein the random nucleotide fragments as a whole can cover the full length of the large fragment nucleic acid molecule.

5. The method according to any one of claims 1-4, wherein each of the multiple oligonucleotide tags on the solid matrix contains the same Barcode sequence.

6. The method according to any one of claims 1-5, wherein the capture in step 1) is achieved by partial reverse complementarity between the large fragment nucleic acid molecule and the oligonucleotide tag.

7. The method according to claim 6, wherein the capture in step 1) is achieved by reverse complementarity between the 3' end of the large fragment nucleic acid molecule and the 3' end of the oligonucleotide tag.

8. The method according to claim 6 or 7, when the large fragment nucleotide molecule is mRNA, the end of the oligonucleotide tag reverse complementary to the large fragment nucleic acid molecule is polyT.

9. The method according to any one of claims 1-5, wherein the capture in step 1) is carried out by using a capture-assisted oligonucleotide, and both sides of the capture-assisted oligonucleotide are complementary to the oligonucleotide tag and the large fragment nucleic acid molecule respectively.

10. The method according to any one of claims 1-9, in step 2), the single-stranded DNA is prepared by reverse transcription reaction, ligase reaction or DNA polymerase reaction.

11. The method according to claim 10, after the reverse transcription reaction, ligase reaction or DNA polymerase reaction, the double-stranded is denatured to unwind, and only the strand containing the oligonucleotide label and the single-stranded DNA linked to the solid matrix is retained.

12. The method according to any one of claims 1-11, in step 3), the ligation is carried out by a DNA polymerase reaction or a ligase reaction.

13. The method according to claim 12, wherein the ligation is carried out by a ligase reaction.

14. The method according to any one of claims 1-13, in step 4), the extension is carried out by a DNA polymerase reaction.

15. The method according to claim 14, wherein the DNA polymerase reaction is a DNA displacement polymerase reaction.

16. The method according to any one of claims 1-15, after step 4), the double-stranded is denatured to unwind, and only the strand containing the oligonucleotide label and the random nucleotide fragment linked to the solid matrix is retained.

17. The method according to any one of claims 8 and 10-16, each of the multiple oligonucleotide labels on the solid matrix contains a Barcode sequence and a label sequence.

18. The method according to any one of claims 1-17, before the ligation by the first bridging oligonucleotide and the second bridging oligonucleotide in step 3), the 3'-end of the single-stranded DNA end is modified to be reverse complementary to the 3'-end of the oligonucleotide label.

19. The method according to any one of claims 1-18, before the ligation by the first bridging oligonucleotide and the second bridging oligonucleotide in step 3), the oligonucleotide label on the solid matrix that has not captured the large fragment nucleic acid molecule is modified so that its 3'-end carries a tag sequence; and the 3'-end of the single-stranded DNA end is modified to be at least partially reverse complementary to the tag sequence; and the tag sequence is at least partially reverse complementary to the remaining 3'-portion of the first bridging oligonucleotide.

20. A method for constructing a sequencing library, comprising: amplifying using the random nucleotide fragment of the large fragment nucleic acid molecule carried on the solid matrix prepared by the method according to any one of claims 1-19 as a template to obtain an amplification product containing the oligonucleotide label and the random nucleotide fragment; and adding a sequencing adapter to the amplification product.

21. A sequencing library prepared by the method according to claim 20.

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