HK1223660B - Sequencing library construction method, and kit and application thereof - Google Patents

Description

A method for constructing a sequencing library, a kit and its application

Technical Field

The present invention relates to the field of sequencing technology, and in particular to a method for constructing a sequencing library, a kit and applications thereof.

Background Art

Next-generation sequencing technology has been extremely popular since its introduction due to its high throughput and low cost. With the development of technology, next-generation sequencing technology has been applied in many scientific research and clinical testing areas.

Currently, many scientific research and clinical applications need to be conducted at the single cell level or at the trace level. Analyzing DNA genetic variation information at the single cell level to determine whether a cell, embryo, or individual is diseased or carries a disease gene is also a common research method. For example, preimplantation genetic diagnosis (PGD) in assisted reproductive technology involves performing DNA genetic testing on gametes, single blastomere cells, or embryonic cells to determine whether the genotype of the fertilized egg or embryo is normal and select normal embryos for implantation.

In terms of sequencers, the Ion Proton sequencer developed by Life Technologies uses a new generation of semiconductor sequencing technology and has been widely welcomed for its advantages of miniaturization and speed.

In the actual use of the Ion Proton sequencer to complete single-cell detection of chromosomal nucleation abnormalities (CNV), timeliness is often very demanding. For example, in preimplantation diagnosis in assisted reproductive technology, if the embryos are not frozen, it takes 24 hours to obtain the test results. This requires shortening the time as much as possible at every step of the test.

Construction of a sequencing library is an essential step in detecting single-cell chromosome number abnormalities through sequencing. Traditional sequencing library construction methods primarily involve mechanically fragmenting target DNA, such as genomic DNA, using a fragmentation instrument (such as the Covaris), followed by end repair and adapter addition (as shown in the PF library construction process on the left in Figure 1). Mechanically fragmented fragments exhibit high randomness, but throughput also relies on a large number of Covaris fragmentation instruments, requiring subsequent separate end processing, adapter addition, PCR, and various purification operations.

Methods for constructing sequencing libraries by simultaneously fragmenting DNA and adding adapters using transposases have been reported. For example, International Patent Application WO2010/048605 discloses a transposon end composition and method for constructing sequencing libraries, which can reduce sample processing time. However, because DNA fragmentation and adapter addition achieved by transposases only add tags to the 5' end of the target DNA, a further step, catalyzed by a nucleic acid-modifying enzyme such as a DNA polymerase, is required to add a tag sequence to the 3' end to produce a double-adapted DNA library. This inevitably increases the time required for library construction.

Summary of the Invention

The present invention provides a method for constructing a sequencing library, a kit, and applications thereof. The sequencing library construction method uses a transposase to achieve a one-step DNA fragmentation and adds sequencing adapters to the 5' and 3' ends, respectively. The sequencing adapters contain sample label information, and can simultaneously sequence samples from different sources, further realizing the detection of abnormal chromosome numbers. Compared with conventional library construction methods, this method saves time, is simple to operate, and has lower requirements for experimental equipment and reaction conditions, thus facilitating the promotion and application of next-generation sequencing technology for detecting single cells or trace amounts of DNA.

According to a first aspect of the present invention, a method for constructing a sequencing library is provided, comprising: incubating target DNA with a transposase-embedded complex under transposition reaction conditions to generate a DNA library with double adapters at both ends; wherein the transposase-embedded complex comprises a transposase, a sequence complementary to a transposase recognition sequence, a first sequencing adapter sequence, and a second sequencing adapter sequence, wherein the first sequencing adapter sequence comprises a first sequencing tag sequence at the 5' end and a transposase recognition sequence at the 3' end, and the second sequencing adapter sequence comprises a second sequencing tag sequence at the 5' end, a sample tag sequence, and a transposase recognition sequence at the 3' end.

The target DNA used in the present invention can be genomic DNA or amplified DNA, such as whole genome amplified DNA. The genomic DNA sample can be derived from a single human cell, a small number of cells, or a trace DNA sample. The cell type can include embryonic cells for preimplantation genetic testing, single tumor cells for cancer research, maternal peripheral blood nucleated red blood cells, plasma, amniotic fluid for prenatal diagnosis, or tissue sections for pathological studies.

In the present invention, whole genome amplification refers to whole genome amplification of a single cell, several cells or a trace nucleic acid sample, and the method can be any of the methods such as partial random primer amplification (Degenerate Oligonucleotide Primer PCR, abbreviated as DOP-PCR), complete random primer amplification (Primer Extension Preamplification PCR, abbreviated as PEP-PCR), multiple strand displacement amplification (Multiple Displacement Amplification, abbreviated as MDA), OmniPlex WGA, etc. Commercial kits such as QIAgen's REPLI-g, SigmaAldrich's GenomePlex WGA, New England Biolabs' Sureplex, RubiconGenomics' PicoPlex WGA, GE Healthcare's illustra Genomiphi V2, etc. can also be used.

The method of the present invention can perform chromosome copy number analysis on sequencing sequences generated by a new generation of high-throughput semiconductor sequencing platforms, including but not limited to the IonTorrent ^™ and Ion Proton ^™ sequencing platforms.

In the present invention, the sample tag sequence is a random sequence, preferably a random sequence of 6-14 bases, more preferably a random sequence of 10 bases. Since each site of the random sequence has four options: A, T, C and G, theoretically, if the random sequence has N bases, 4 ^N sample tag sequences can be generated. Therefore, a random sequence of 10 bases is sufficient to label the sequencing sample.

As a preferred technical solution of the present invention, the second sequencing adapter sequence also includes the sequencing-specific base "GAT" between the sample tag sequence and the transposase recognition sequence at the 3' end. Adding three bases "GAT" after the sample tag sequence avoids two consecutive Cs, which can cause tag recognition errors during subsequent analysis.

As a preferred technical solution of the present invention, the first sequencing tag sequence and/or the second sequencing tag sequence are selected from tag sequences of the Ion Torrent ^™ or Ion Proton ^™ sequencing platforms; therefore, the method of the present invention is applicable to the IonTorrent ^™ or Ion Proton ^™ sequencing platforms.

As a preferred technical solution of the present invention, the transposase recognition sequence is a 19 bp chimeric transposon end recognized by transposase Tn5.

As a preferred technical solution of the present invention, the transposase recognition sequence complementary sequence has the base sequence shown in SEQ ID NO: 1; the first sequencing adapter sequence has the base sequence shown in SEQ ID NO: 2; and the second sequencing adapter sequence has the base sequence shown in SEQ ID NO: 3.

SEQ ID NO: 1 is 5'-CTGTCTCTTATACACATCT-3'. It should be noted that the complementary sequence of the transposase recognition sequence of the present invention is not limited to the base sequence shown in SEQ ID NO: 1, and there may be several additional base sequences at both the 5' end and the 3' end. SEQ ID NO: 2 is:

5'-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT AGATGTGTATAAGAGACAG -3'; wherein the underlined portion is the transposase recognition sequence, and the non-underlined portion is the first sequencing tag sequence. It should be noted that the first sequencing adapter sequence of the present invention is not limited to the base sequence shown in SEQ ID NO: 2. Several additional base sequences or linker sequences may be present before, after, and between the transposase recognition sequence and the first sequencing tag sequence. SEQ ID NO: 3 is:

5'-CCATCTCATCCCTGCGTGTCTCCGACTCAGNNNNNNNNNNGAT AGATGTGTATAAGAGACAG -3'; wherein the underlined portion is the transposase recognition sequence, NNNNNNNNNN is the sample tag sequence, each N can be selected from any of A, T, C, and G, the sequence before NNNNNNNNNN is the second sequencing tag sequence, and the subsequent GAT is a special sequencing base. It should be noted that the second sequencing adapter sequence of the present invention is not limited to the base sequence shown in SEQ ID NO: 3. Several additional base sequences or linker sequences may be present before and/or after the transposase recognition sequence, and several additional base sequences may be present before the second sequencing tag sequence.

According to a second aspect of the present invention, the present invention provides a kit for constructing a sequencing library, the kit comprising a transposase recognition sequence complementary sequence, a first sequencing adapter sequence and a second sequencing adapter sequence, wherein the first sequencing adapter sequence comprises a first sequencing tag sequence at the 5' end and a transposase recognition sequence at the 3' end, and the second sequencing adapter sequence comprises a second sequencing tag sequence at the 5' end, a sample tag sequence and a transposase recognition sequence at the 3' end.

As a preferred technical solution of the present invention, the sample tag sequence is a random sequence, preferably a random sequence of 6-14 bases, more preferably a random sequence of 10 bases.

As a preferred technical solution of the present invention, the second sequencing adapter sequence further includes a sequencing-specific base "GAT" between the sample tag sequence and the transposase recognition sequence at the 3' end.

As a preferred technical solution of the present invention, the first sequencing tag sequence and/or the second sequencing tag sequence are selected from tag sequences of Ion Torrent ^™ or Ion Proton ^™ sequencing platforms.

As a preferred technical solution of the present invention, the transposase recognition sequence is a 19 bp chimeric transposon end recognized by transposase Tn5.

As a preferred technical solution of the present invention, the transposase recognition sequence complementary sequence has the base sequence shown in SEQ ID NO: 1; the first sequencing adapter sequence has the base sequence shown in SEQ ID NO: 2; and the second sequencing adapter sequence has the base sequence shown in SEQ ID NO: 3.

As a preferred technical solution of the present invention, the kit further comprises a transposase, and the transposase is preferably transposase Tn5. In a specific embodiment of the present invention, Tagment Enzyme from Vazyme is used, but other transposases of this type are also applicable to the present invention.

As a preferred embodiment of the present invention, the kit also includes a DNA polymerase for nick translation. In one embodiment, Life Technologies' Platinum Pfx DNA polymerase is used, but other DNA polymerases are also suitable. The DNA polymerase can fill in the DNA nicks created by the transposase through nick translation, facilitating subsequent sequencing.

The description in the first aspect also applies to the second aspect. There is no substantial difference between the two, so I will not go into details here.

It should be noted that the concepts of "first" and "second" in the present invention are only used to distinguish different objects of expression and can be understood as having technical meanings or meanings with sequential limitations.

According to the third aspect of the present invention, the present invention provides the use of the kit as described in the second aspect in the construction of a sequencing library and the detection of abnormal chromosome numbers by sequencing, preferably in the detection of abnormal chromosome numbers in single cells.

Compared with the existing technology, the advantages of the present invention are reflected in: the method for constructing the sequencing library of the present invention uses a transposase recognition sequence complementary sequence, a first sequencing adapter sequence and a second sequencing adapter sequence, wherein the second sequencing adapter sequence contains a special sample label sequence as the label information of the sample, and the use of transposase can achieve a one-step DNA shearing method and add different sequencing adapters at the 5' end and 3' end at the same time, without the need for DNA polymerase and other nucleic acid modification enzymes to catalyze the addition of 3' end label sequences as in the existing transposase-based DNA shearing method. The sequencing adapter of the present invention contains sample label information, which can realize the sequencing of samples from different sources at the same time, and further realize the detection of abnormal chromosome numbers. The method of the present invention saves time compared to conventional library construction methods, can save nearly 5 hours of library construction time, and is simple to operate, has low requirements for experimental equipment and reaction conditions, and is conducive to the promotion and application of next-generation sequencing detection of single cells or trace DNA technology.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a comparison diagram of the traditional PF library construction process (left) and the transposase library construction process of the present invention (right).

FIG2 is a schematic diagram showing the principle of using transposase to complete DNA fragmentation and sequencing adapter ligation in one step according to the present invention.

FIG3 is a comparison of the karyotype diagram (a and b) and the result peak diagram (c and d) obtained by sequencing analysis of the sample S1 in the present invention using the method of the present invention (a and c) and the conventional library construction method (b and d).

Figure 4 is a comparison of the karyotype diagram (a and b) and the result peak diagram (c and d) obtained by sequencing analysis of sample S2 in the present invention using the conventional library construction method (a and c) and the method of the present invention (b and d).

DETAILED DESCRIPTION

The present invention will be further described in detail below through specific embodiments with reference to the accompanying drawings.

As shown in Figure 1, the traditional PF library construction process (left figure in Figure 1) includes steps such as DNA fragmentation by Covaris fragmentation instrument, end repair, adapter addition, quality inspection, library pooling, gap translation, re-quality inspection, and loading; while the transposase library construction process of the present invention (right figure in Figure 1) includes steps such as transposition reaction mixture preparation, transposition reaction (DNA fragmentation and adapter addition), quality inspection, library pooling, gap translation, re-quality inspection, and loading. It can be seen that the transposition reaction of the present invention replaces the three steps of DNA fragmentation, end repair, and adapter addition by the traditional Covaris fragmentation instrument in one step, which significantly saves time.

As shown in Figure 2, the principle of the present invention using transposase to complete DNA shearing and sequencing adapter ligation in one step is as follows: the transposase recognition sequence-reverse (ME-r, i.e., the transposase recognition sequence complementary sequence) is annealed with sequencing adapter sequence 1 (i.e., the first sequencing adapter sequence) and tagged sequencing adapter sequence 2 (i.e., the second sequencing adapter sequence, where the tag is the sample tag sequence) to form adapters, which are then embedded with the transposase to form a transposase-embedded complex. The transposase-embedded complex is then incubated with genomic DNA or amplified products for transposition shearing to obtain DNA fragments with double adapters at both ends. A DNA library is obtained by extension (nick translation reaction); and single-stranded DNA is then generated by emulsion PCR for sequencing.

The present invention is described in detail below through specific embodiments.

1. Sample selection and whole genome amplification

Eight human lymphocyte cell lines with known karyotypes were selected, including samples with aneuploidy and deletions/duplications of varying sizes (the smallest of which was approximately 1.9 Mb). When these cells reached optimal growth, single cells or cell clusters were isolated for whole-genome amplification, and DNA quantification was performed using a Nanodrop spectrophotometer. Two whole-genome amplification kits, GenomePlex WGA from Sigma Aldrich and Sureplex from New England Biolabs, were used in parallel for amplification. Both single-cell and multi-cell groups were used for each cell line, resulting in a total of 32 whole-genome amplification product samples.

100 ng of DNA was collected from each sample to complete the construction of the transposase library of the present invention.

In addition, 100 ng of DNA was collected from each sample and constructed using the standard library construction method published on the official website of Life Technologies as a control. For detailed procedures, please refer to the official website of Life Technologies (http://www.lifetechnologies.com).

2. Joint preparation

Synthesize the following linker:

ME-r: 5'-CTGTCTCTTATACACATCT-3' (SEQ ID NO: 4);

P1: 5'-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT

AGATGTGTATAAGAGACAG-3' (SEQ ID NO: 5);

PA_1: 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG CTAAGGTA

AC GATAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 6);

PA_2: 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG TAAGGAGA

AC GATAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 7);

PA_3: 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG AAGAGGAT

TC GATAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 8);

PA_4: 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG TACCAAGA

TC GATAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 9);

PA_5: 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG CAGAAGGA

AC GATAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 10);

PA_6: 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG CTGCAAGT

TC GATAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 11);

PA_7: 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG TTCGTGAT

TC GATAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 12);

PA_8: 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG TTCCGATA

AC GATAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 13).

Note: The underlined part is the sample tag sequence. In the reaction, different samples are selected with different tags PA_N to distinguish them.

ME-r, P1, and PA_1-8 were dissolved to 100 μM using annealing buffer.

Note: Annealing buffer is prepared as follows: accurately weigh 1.21 g Tris-base (100 mM), 5.844 g NaCl (1000 mM), and 0.372 g EDTA ₂ Na (10 mM), add ultrapure water to a final volume of 100 mL, fully dissolve, and mix well to prepare 10× annealing buffer.

Prepare the reaction system in a 200 μL PCR tube according to the following table (Table 1):

Table 1

Vortex and mix thoroughly, then briefly centrifuge each of the nine prepared aliquots from reactions 1 and 2 (1-8). Place the aliquots in a PCR instrument and complete the reaction according to the following protocol (Table 2):

Table 2

After the reaction is completed, equal volumes of 1 to 8 of reaction 1 and reaction 2 are mixed and homogenized, and the mixtures are named "annealing linker mixtures 1 to 8" and stored at -20°C.

3. Annealing adapter mixture-transposase embedding

Prepare the reaction system in eight 200 μL PCR tubes according to the following table (Table 3):

Table 3

Note: The transposase used in the examples is Tagment Enzyme produced by Vazyme, with a specification of (10U/μL); the embedding buffer is a supporting reagent for the transposase, also produced by Vazyme.

Mix thoroughly by pipetting gently at least 20 times.

The prepared reaction system was placed on a PCR instrument at 30°C for 1 hour. The reaction products were named "Transposition Reaction Mixtures 1 to 8" and stored at -20°C.

4. DNA fragmentation and addition of sequencing adapters

Thaw the transposition reaction buffer at room temperature, mix by inverting the tube until ready for use.

Prepare the following reaction systems (Table 4) in 8 PCR tubes respectively:

Table 4

Note: Transposition reaction buffer is a supporting reagent for transposase, also produced by Vazyme.

Mix thoroughly by pipetting gently at least 20 times.

Place the mixed reaction system on a PCR instrument and perform the reaction according to the following procedure (Table 5):

Table 5

After the reaction is completed, remove the PCR tube, purify with 1.5 times the volume of Ampure XP Beads, and dissolve in 25 μL of EB.

5. Library Pooling

Take 3 μL of each of the 8 products obtained in the previous step and mix them in equal volumes to obtain 24 μL of a mixed solution.

6. Cut translation

Prepare the following reaction system in a PCR tube (Table 6):

Table 6

Note: The amplification enzyme is Platinum Pfx DNA polymerase from Life Technologies, and the amplification buffer is the matching reagent.

Mix thoroughly by pipetting gently 10 times.

The mixed reaction system was placed on a PCR instrument and incubated at 72°C for 20 min.

After the reaction is completed, remove the PCR tube, purify with 1.2 times the volume of Ampure XP Beads, and dissolve in 16 μL of EB.

7. Sequencing

After the product passed the library test, it was sequenced using the Ion Proton ^™ sequencing platform.

8. Post-sequencing information analysis

The data obtained from sequencing the 8 samples using the above process, together with the 8 sequencing data obtained from conventional library construction, were analyzed according to the following process:

1) Extract valid data: Convert the bam format off-machine data to the fastQ format required by the alignment software, and cut off 50 bp from the 5' end of the read for subsequent analysis. On this basis, cut off another 20 bp from the 5' end to eliminate the influence of the adapter introduced during whole genome amplification (WGA) on subsequent analysis;

2) Sequence alignment: The extracted reads were aligned with the human genome reference sequence version 37.3 (hg19; NCBIBuild37.3) in the NCBI database using SOAPaligner/soap2;

3) Y chromosome determination: Determine whether the Y chromosome exists based on the support number of Y chromosome-specific genes;

4) Window division: The human genome reference sequence is divided into windows of approximately 100 kb, and the windows are slid up and down by 20 kb;

5) GC content correction: Count the number of unique reads in each window (i.e., sequences with only one alignment position on the reference genome in the deduplicated sequence) and calculate their GC content (GC%). The median GC% of the reads in each window is used as the GC% of the window. Each window on the sample sequence and the reference sequence is divided into different correction units according to GC% (with a gradient of 0.05), and the median (Mi) of the number of reads in different windows within each correction unit is calculated. This is used to calculate the correction coefficient of each correction unit, and then the corrected Ratio value of each window is calculated for subsequent analysis;

6) Breakpoint screening: Treat each window as a point and perform a run test on each point to obtain a preliminary breakpoint set. Then, perform multiple screening on the points in the breakpoint set to determine the final breakpoint set.

7) Data Filtering and Visualization: In this method, a positive CNV signal must meet three conditions: a) CNV fragment size is not less than 1M; b) P ≤ 1e-10; c) Ratio ≤ 0.7 (deletion) or Ratio ≥ 1.3 (duplication). CNV is determined based on these conditions, and the corresponding karyotype and peak plot corresponding to the ratio value in each window are plotted.

9. Results Analysis

The results obtained by the above analysis are shown in the following table (Table 7). A total of 8 samples were tested this time. The results of the detection method of the present invention were compared with the known results and the results obtained by conventional library construction, and the results were completely consistent.

Table 7

Figure 3 shows a comparison of the karyotypes and peak plots obtained for sample S1 using the present invention's method and conventional library construction methods. Figure 3a shows the karyotype obtained using the present invention's method; Figure 3b shows the karyotype obtained using the conventional library construction method; Figure 3c shows the peak plot obtained using the present invention's method; and Figure 3d shows the peak plot obtained using the conventional library construction method.

Figure 4 shows a comparison of the karyotypes and peak plots obtained for sample S2 using conventional library construction and sequencing analysis using the method of the present invention. Figure 4a shows the karyotype obtained using the conventional library construction method; Figure 4b shows the karyotype obtained using the method of the present invention; Figure 4c shows the peak plot obtained using the conventional library construction method; and Figure 4d shows the peak plot obtained using the method of the present invention.

The results shown in Figures 3 and 4 demonstrate that the results obtained by the method of the present invention are completely consistent with those obtained by conventional library construction methods, indicating that the method of the present invention can greatly simplify the library construction process and shorten the library construction time while ensuring the authenticity of the results.

The above is a further detailed description of the present invention in conjunction with specific embodiments, and the specific implementation of the present invention cannot be considered to be limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention.

Claims (21)

1. A method for constructing a sequencing library, characterized in that the method comprises: incubating target DNA with a transposase embedding complex under transposition reaction conditions to generate a DNA library with dual adapters at both ends; wherein the transposase embedding complex comprises a transposase, a transposase recognition sequence complementary sequence, a first sequencing adapter sequence, and a second sequencing adapter sequence, the first sequencing adapter sequence comprising a first sequencing tag sequence at the 5' end and a transposase recognition sequence at the 3' end, and the second sequencing adapter sequence comprising a second sequencing tag sequence at the 5' end, a sample tag sequence, and a transposase recognition sequence at the 3' end. 2. The method according to claim 1, wherein the sample label sequence is a random sequence. 3. The method according to claim 1, wherein the sample tag sequence is a random sequence of 6-14 bases. 4. The method according to claim 1, wherein the sample tag sequence is a random sequence of 10 bases. 5. The method according to claim 1, wherein the second sequencing adapter sequence further includes a sequencing-specific base “GAT” between the sample tag sequence and the transposase recognition sequence at the 3’ end. 6. The method according to claim 1, wherein the first sequencing tag sequence and/or the second sequencing tag sequence are selected from tag sequences of the Ion Torrent ^™ or Ion Proton ^™ sequencing platforms. 7. The method according to claim 1, wherein the transposase recognition sequence is the 19bp chimeric transposon end recognized by transposase Tn5. 8. The method according to claim 1, wherein the complementary sequence of the transposase recognition sequence has the base sequence shown in SEQ ID NO: 1; the first sequencing adapter sequence has the base sequence shown in SEQ ID NO: 2; and the second sequencing adapter sequence has the base sequence shown in SEQ ID NO: 3. 9. A kit for constructing a sequencing library, characterized in that the kit comprises a transposase recognition sequence complementary sequence, a first sequencing adapter sequence, and a second sequencing adapter sequence, wherein the first sequencing adapter sequence comprises a first sequencing tag sequence at the 5' end and a transposase recognition sequence at the 3' end, and the second sequencing adapter sequence comprises a second sequencing tag sequence at the 5' end, a sample tag sequence, and a transposase recognition sequence at the 3' end. 10. The kit according to claim 9, wherein the sample label sequence is a random sequence. 11. The kit according to claim 9, wherein the sample tag sequence is a random sequence of 6-14 bases. 12. The kit according to claim 9, wherein the sample tag sequence is a random sequence of 10 bases. 13. The kit according to claim 9, wherein the second sequencing adapter sequence further includes a sequencing-specific base “GAT” between the sample tag sequence and the transposase recognition sequence at the 3’ end. 14. The kit according to claim 9, wherein the first sequencing tag sequence and/or the second sequencing tag sequence are selected from tag sequences of the Ion Torrent ^™ or Ion Proton ^™ sequencing platforms. 15. The kit according to claim 9, wherein the transposase recognition sequence is the 19bp chimeric transposon terminus recognized by transposase Tn5. 16. The kit according to claim 9, wherein the complementary sequence of the transposase recognition sequence has the base sequence shown in SEQ ID NO: 1; the first sequencing adapter sequence has the base sequence shown in SEQ ID NO: 2; and the second sequencing adapter sequence has the base sequence shown in SEQ ID NO: 3. 17. The kit according to claim 9, wherein the kit further comprises a transposase. 18. The kit according to claim 17, wherein the transposase is transposase Tn5. 19. The kit according to claim 9, wherein the kit further comprises a DNA polymerase for the nick translation reaction. 20. The application of the kit according to any one of claims 9-19 in the construction of sequencing libraries and the detection of chromosome number abnormalities by sequencing. 21. The application of the kit according to any one of claims 9-19 in the construction of sequencing libraries and the detection of single-cell chromosome number abnormalities by sequencing.