A method and application for analyzing structural variations based on third-generation single-molecule sequencing

Abstract

This invention discloses a method and application for analyzing structural variations based on third-generation single-molecule sequencing. The method includes: fixing and cross-linking the sample to obtain fixed cells; lysing and permeabilizing the fixed cells; releasing chromatin; breaking the chromatin to initiate a ligation reaction to obtain ligation products; decross-linking the ligation products to obtain DNA products; constructing a third-generation single-molecule sequencing library from the DNA products to obtain a sequencing library; performing third-generation single-molecule sequencing on the sequencing library to obtain sequencing data; and performing bioinformatics analysis on the sequencing data to analyze the structural variations of the sample. This method effectively reduces blood collection volume, cell input, and reagent consumption. Compared with second-generation high-throughput chromatin conformation capture methods, it has significant advantages and performance improvements in long-range signal coverage, SNP count, repetitive sequence coverage, centromere coverage, and coverage of highly homologous sequences.

Description

Technical Field

[0001] This invention belongs to the field of chromosome structural variation detection technology, and relates to a method and application for analyzing structural variations based on third-generation single-molecule sequencing. Background Technology

[0002] Genomic structural variations (SVs), including deletions, duplications, inversions, and translocations, play a crucial role in the evolution of hereditary diseases and cancer genomes, as well as population genetic diversity. Traditional structural variation detection primarily relies on next-generation sequencing (NGS) technology, inferring variations by analyzing anomalous signals in read alignments (such as changes in coverage or abnormal alignment orientation). However, conventional NGS methods have limitations in detecting complex structural variations, accurately locating breakpoints, and distinguishing haplotype phase information.

[0003] In recent years, three-dimensional genomics technologies, especially high-throughput chromatin conformation capture (Hi-C) technology, have effectively revealed long-range chromatin interactions across the entire genome. Studies have found that chromatin spatial interaction maps exhibit unique pattern changes in genomic regions with structural variations, providing new bioinformatics features for the detection and analysis of structural variations. However, next-generation sequencing inherently has short read lengths (typically 100-300 bp), which significantly limits its detection capabilities. First, in heterochromatin, repetitive sequences, or highly homologous regions, short reads are difficult to uniquely align, leading to significant data loss and coverage, insufficient resolution, and difficulty in accurately locating breakpoints. Second, the process of linking paired-end reads to infer haplotype information is relatively indirect and inefficient.

[0004] Therefore, there is an urgent need for a new method or system that can integrate the advantages of chromosome conformation technology and third-generation single-molecule sequencing technology, while ensuring detection accuracy and resolution, and improving its economy and analytical robustness, so as to promote the widespread application of three-dimensional genomics in the field of structural variation detection. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, this invention provides a method and application for analyzing structural variations based on third-generation single-molecule sequencing. This method solves the problems of insufficient resolution, difficulty in accurately locating breakpoints, inability to meet the needs of detecting complex structural variations in clinical settings, long detection time, low efficiency, and difficulty in constructing haplotypes in existing technologies.

[0006] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for analyzing structural variations based on third-generation single-molecule sequencing. The method includes: fixing and cross-linking a sample to be tested to obtain fixed cells; lysing and permeabilizing the fixed cells; releasing chromatin; using restriction endonucleases to break down the chromatin and perform a ligation reaction to obtain a ligation product; decross-linking the ligation product to obtain a DNA product; constructing a third-generation single-molecule sequencing library from the DNA product to obtain a sequencing library; performing third-generation single-molecule sequencing on the sequencing library to obtain sequencing data; and performing bioinformatics analysis on the sequencing data to analyze the structural variations of the sample. The number of cells in the sample to be tested is 0.01 × 10⁻⁶. 6 -100×10 6 indivual.

[0007] This invention effectively reduces blood collection volume, cell input, and reagent usage, shortening the operation time from 4-5 days to 2 days, and eliminating the need for overnight waiting. Compared with second-generation high-throughput chromatin conformation capture methods, this invention offers significant advantages and performance improvements in long-range signal coverage, SNP count, repetitive sequences, centromere coverage, and coverage of highly homologous sequences.

[0008] Preferably, the cell count of the sample to be tested is 0.01 × 10⁻⁶. 6 -100×10 6 One, for example, 0.01 × 10 6 1×10 6 10×10 6 One or 100×10 6 indivual.

[0009] Preferably, the sample to be tested includes any one or a combination of at least two of the following: blood, blood products, amniotic fluid, chorionic villus tissue, or isolated cells.

[0010] Preferably, the source of the isolated cells includes any one or a combination of at least two of lymphocytes, stem cells, or bone marrow.

[0011] Preferably, the sample to be tested is peripheral blood mononuclear cells.

[0012] Preferably, the lysis and permeabilization treatment includes: lysing the fixed cells using lysis buffer and protease inhibitor, and then adding sodium dodecyl sulfate and Triton X-100 for permeabilization treatment; the total volume of the lysis and permeabilization treatment system is 100-160 μL, for example 120 μL, 130 μL, 140 μL or 146 μL.

[0013] Preferably, the restriction endonuclease includes any one or a combination of at least two of DpnII, HinfI, or DdeI.

[0014] Preferably, the chromatin breaking conditions are 35-38℃ (e.g., 35℃, 37℃ or 38℃) with shaking incubation for 0.5-2 h (e.g., 0.5 h, 1 h or 2 h).

[0015] Preferably, the total volume of the reaction system for disrupting chromatin is 145-150 μL, for example, 145 μL, 146 μL, 148 μL or 150 μL.

[0016] Preferably, the ligation reaction system includes T4 DNA ligase and ligation buffer.

[0017] Preferably, the amount of T4 DNA ligase used is 2-5 μL / reaction, for example, 2 μL / reaction, 4 μL / reaction or 5 μL / reaction.

[0018] Preferably, the ligation buffer contains T4 DNA ligase buffer and polyethylene glycol.

[0019] Preferably, the temperature of the ligation reaction is 20-25°C (e.g., 20°C, 23°C or 25°C), and the time of the ligation reaction is 0.5-2 h (e.g., 0.5 h, 1 h or 2 h).

[0020] Preferably, the decrosslinking treatment includes enzymatic hydrolysis using proteinase K and / or streptomycin, followed by shaking incubation at 58-62°C (e.g., 58°C, 60°C, or 62°C) for 0.5-2 h (e.g., 0.5 h, 1 h, or 2 h).

[0021] Preferably, the total volume of the decrosslinking treatment system is 120-200 μL, for example, 160 μL, 163 μL or 165 μL.

[0022] Preferably, the construction of the third-generation single-molecule sequencing library includes the construction of a nanopore sequencing library.

[0023] Preferably, the bioinformatics analysis includes: aligning sequencing data to a reference genome to obtain an alignment file; identifying enzyme restriction sites to obtain re-alignment results; generating QC quality control information; performing SNP / INDEL variant analysis; generating an interaction signal file; and identifying structural variants to obtain structural variant breakpoint information.

[0024] Preferably, the identification of structural variations includes: establishing an interaction signal database of normal samples, removing background noise signals from the samples to be tested, and obtaining accurate structural variation signals.

[0025] Preferably, the bioinformatics analysis further includes copy number variation identification and / or SNP haplotype construction of chromosomes with structural variations.

[0026] Preferably, the copy number variation identification includes: establishing a copy number reference database for the whole genome of normal samples, calculating the copy number results of the sample to be tested, generating the Log value of the chromosome window, and determining the breakpoint of the copy number difference.

[0027] Preferably, the construction of the SNP haplotype of the structurally variable chromosome includes the following steps: (1) Sequence assembly of the target chromosome was performed to obtain SNP information of the Hap1 haplotype and Hap2 haplotype of the chromosome; (2) Identify the breakpoint of the target structural variation and extract the sequence information of the chromosome where the structural variation is located; (3) Extract sequences that conform to the structural variation characteristics in the upstream and downstream regions of the breakpoint, analyze their variation, and obtain haplotype bases carrying chromosomes and haplotype bases of normal chromosomes. (4) Extend upstream and downstream of the fracture point to obtain structural variation monomer type SNP information within the upstream and downstream range of the fracture point; (5) Match and extend the monomeric SNP information obtained in step (4) with the monomeric SNP information obtained in step (1) to obtain complete structural variation monomeric SNP information.

[0028] As a preferred technical solution, the method for analyzing structural variations based on third-generation single-molecule sequencing according to the present invention includes the following steps: (1) Peripheral blood mononuclear cells of the test sample were fixed and cross-linked with formaldehyde to obtain fixed cells; (2) The fixed cells were lysed using lysis buffer and protease inhibitor, and then permeabilized with SDS and Triton X-100 to release chromatin; (3) Use restriction endonuclease DpnII to break down chromatin; (4) The fragmented chromatin was ligated using T4 DNA ligase in a ligation buffer containing PEG. The mixture was then incubated with shaking at 23°C to obtain the ligation product. (5) The ligation product is decrosslinked, enzymatically digested with proteinase K and / or streptomycin, and purified using magnetic beads to obtain DNA product; (6) Construct a third-generation single-molecule sequencing library from the DNA product to obtain the sequencing library; (7) Perform third-generation single-molecule sequencing on the sequencing library to obtain sequencing data; (8) Align the sequencing data to the reference genome, obtain the alignment file, identify the restriction enzyme sites, obtain the re-alignment results, generate QC quality control information, perform SNP / INDEL variant analysis, generate the interaction signal file, identify structural variants, and obtain structural variant breakpoint information.

[0029] Secondly, the present invention provides the application of the method for analyzing structural variations based on third-generation single-molecule sequencing described in the first aspect in the preparation of products for genetic disease screening, preconception screening, tumor research, or structural variation detection.

[0030] Preferably, the genetic disease screening includes screening for non-carrier embryos.

[0031] Thirdly, the present invention provides a detection device for implementing the method for analyzing structural variations based on third-generation single-molecule sequencing as described in the first aspect, the detection device comprising a cell extraction and fixation module, a chromosome conformation capture module, a sequencing library construction module, a sequencing module, a structural variation analysis module, and a haplotype construction module; The cell extraction and fixation module is used to perform the following: extracting cells from a sample and fixing them into a conformation; The chromosome conformation capture module is used to perform actions including: cell lysis and permeation, chromatin fragmentation, ligation, and decrosslinking. The sequencing library construction module is used to perform actions including: constructing a nanopore sequencing library; The sequencing module is used to perform the following operations: sequencing a nanopore sequencing library, aligning the sequencing data to a reference genome to obtain an alignment file; identifying restriction enzyme sites to obtain re-alignment results; generating QC quality control information; performing SNP / INDEL variant analysis; and generating an interaction signal file. The structural variation analysis module is used to perform the following tasks: establishing an interactive signal database for normal samples, removing background noise signals from the samples to be tested, and obtaining accurate structural variation signals. The haplotype construction module is used to perform the following: sequence assembly of the target chromosome, obtaining SNP information of the Hap1 and Hap2 haplotypes of the chromosome, identifying the breakpoint of the target structural variation, extracting the sequence information of the chromosome where the structural variation is located, extracting sequences that conform to the characteristics of the structural variation in the upstream and downstream regions of the breakpoint, analyzing their variation, obtaining the haplotype bases carrying the chromosome and the haplotype bases of the normal chromosome, extending upstream and downstream of the breakpoint to obtain the structural variation haplotype SNP information within the upstream and downstream range of the breakpoint, and matching and extending the obtained haplotype SNP information to obtain complete structural variation haplotype SNP information.

[0032] Compared with the prior art, the present invention has the following beneficial effects: (1) The method for analyzing structural variations based on third-generation single-molecule sequencing described in this invention can effectively reduce the amount of blood collected (5 mL vs 2 mL), the amount of cells input (10 M vs 1 M), and the amount of reagents compared with conventional methods. The operation time is shortened from 4-5 days to 2 days, and there is no need for overnight waiting and operation. (2) The method of the present invention can effectively cover complex repetitive sequence regions, centromeres, and highly homologous regions, thereby improving detection resolution; (3) The method of the present invention can effectively construct haplotypes, increase the number of effective SNPs, and can be used more accurately for embryo non-carrier screening. Attached Figure Description

[0033] Figure 1 This is a spatial contact map of chromosome 9 obtained using the method of the present invention.

[0034] Figure 2 This is a spatial contact map of chromosome 9 obtained using a second-generation high-throughput chromatin conformation capture method.

[0035] Figure 3 This is a spatial contact map of chromosome 16 obtained using the method of the present invention.

[0036] Figure 4 This is a spatial contact map of chromosome 16 obtained using a second-generation high-throughput chromatin conformation capture method.

[0037] Figure 5 This is a haplotype analysis map of the embryo obtained by using the method of the present invention.

[0038] Figure 6 This is an analysis map of embryonic haplotypes obtained using a second-generation high-throughput chromatin conformation capture method. Detailed Implementation

[0039] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0040] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0041] To facilitate a clearer understanding of this invention, certain terms are first defined. As used herein, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All patents and publications mentioned herein are incorporated herein by reference in their entirety.

[0042] The term "PBMC" refers to peripheral blood mononuclear cells.

[0043] The term "Hi-C technology" refers to chromosome conformation capture technology.

[0044] The term "spatial contact matrix" refers to the chromosome interaction matrix or contact matrix, which quantifies the frequency of contact between different locations on chromatin in the three-dimensional space of the cell nucleus, and intuitively reveals the spatial conformation and interaction of chromatin.

[0045] The term "Structural Variations (SVs)" refers to genomic structural variations, including deletions, duplications, inversions, translocations, and other types.

[0046] The term "next generation sequencing (NGS)" refers to next-generation sequencing technology, which is a technology that enables high-speed, low-cost parallel sequencing of millions to billions of DNA fragments.

[0047] The term "Reads" refers to the short sequences generated by a sequencing instrument when it reads a DNA fragment in a single pass; these are the basic data units of high-throughput sequencing.

[0048] The term "third-generation single-molecule sequencing technology" refers to real-time, long-read sequencing of individual DNA molecules without the need for PCR amplification and fragmentation assembly.

[0049] Example 1 This embodiment provides a method for analyzing structural variations based on third-generation single-molecule sequencing, as detailed below: 1. Peripheral blood was collected from each subject in 2 mL cell-free DNA preservation tubes, and PBMC cells were extracted using the cleavage red blood cell method. (1) Take a 15 mL centrifuge tube, add 2 mL of blood from the free DNA preservation tube to the 15 mL centrifuge tube, centrifuge at 500×g for 5 min, and discard the supernatant (yellow plasma part).

[0050] (2) Add 13 mL of red blood cell lysis buffer, invert 10 times, and lyse at room temperature for 10 min. Invert gently every 2 min to mix and promote lysis.

[0051] (3) Centrifuge at 500×g for 5 min and discard the red supernatant.

[0052] (4) Add 6 mL of PBS to resuspend the precipitate, centrifuge at 500×g for 3 min, and discard the supernatant.

[0053] (5) Add 3 mL PBS to resuspend the precipitate, centrifuge at 500×g for 3 min, and discard the supernatant.

[0054] (6) Add 946 μL of 1×PBS buffer to resuspend the mononuclear cells to obtain a cell suspension for subsequent cell fixation.

[0055] (7) Add 54 μL of 37% formaldehyde to 946 μL of cell suspension and quickly invert 10 times to mix thoroughly.

[0056] (8) Incubate at room temperature for 10 min, gently shaking the centrifuge tube every 2 min.

[0057] (9) After incubation at room temperature, add 54 μL of fixation solution, invert 10 times to mix, incubate at room temperature for 5 min, and then incubate on ice for at least 15 min.

[0058] (10) After incubation on ice, centrifuge at 2000×g for 10 min at room temperature and discard the supernatant.

[0059] (11) Add 1 mL of 1×PBS buffer, pipette to resuspend the cells, and obtain 1 mL of resuspended cells.

[0060] Cell counts were performed using a cell counter, and 1×10⁶ cells were collected. 6 Subsequent cell lysis treatment was performed.

[0061] 2. Lysis and permeation of cell membrane and disruption of chromatin (1) Add 20 μL of lysis buffer 1 (10 mM pH8.0 Tris-HCl, 10 mM NaCl, 0.2% IGEPAL) to PBMC cells and resuspend the cells by pipetting.

[0062] (2) Add 1 μL of protease inhibitor, mix by pipetting, incubate on ice for 15 min, and invert and mix every 5 min.

[0063] (3) Add 90 μL of 1× endonuclease buffer and 10 μL of 2% SDS, mix well by pipetting, and incubate at 62℃ for 10 min.

[0064] (4) Add 11 μL of 10% Triton X-100 (1% final concentration), vortex gently to mix, and incubate at 37°C for 10 min.

[0065] (5) After incubation at 37°C, add 5 μL of 10× endonuclease buffer and 20 μL of DpnII, incubate at room temperature at 37°C for 1 h, shake in a metal bath at 850 rpm, then incubate at 65°C for 20 min, and add 250 μL of purified magnetic beads.

[0066] 3. Connection reaction Prepare the bonding mixture as shown in Table 1: Table 1 Add 604 μL of ligation reaction solution to the above enzyme digestion product, vortex to mix (total volume 1 mL), place in a 23°C shaking constant temperature metal bath, and incubate at 850 rpm for 1 h.

[0067] 4. De-crosslinking reaction (1) Take out the magnetic beads and 10% SDS and place them at room temperature for later use. Place proteinase K on an ice box for later use.

[0068] (2) After the ligation incubation is complete, centrifuge at 2500×g at room temperature for 3 min, let stand on a magnetic rack for 2 min, discard the supernatant, and add 150 μL of buffer A to resuspend the ligation product with magnetic beads.

[0069] (3) Add 5 μL of 10% SDS and 8 μL of proteinase K to the product of magnetic bead suspension and vortex mix. Incubate in a metal bath at 60°C with shaking at 850 rpm for 1 h.

[0070] (4) After incubation at 60°C, add 10 μL of pronase (10 mg / mL pronase), place in a constant temperature metal bath at 37°C and incubate at 850 rpm for 1 h.

[0071] (5) Add 85 μL of magnetic beads, vortex to mix, let stand for 5 min, place on a magnetic rack for 2 min, and discard the supernatant.

[0072] (6) Add 200 μL of freshly prepared 80% ethanol to the centrifuge tube, let stand for 30 s, and discard the supernatant.

[0073] (7) Repeat step (5) once.

[0074] (8) Use a small nozzle to remove the residual 80% ethanol, and let stand at room temperature for 2 minutes to dry the magnetic beads.

[0075] (9) Add 24 μL of nuclease-free water, vortex to mix, let stand for 5 min, place on a magnetic rack for 2 min, take 22 μL of supernatant and transfer to a new 0.2 mL centrifuge tube to obtain the decrosslinking product.

[0076] (10) Take 1 μL of the decrosslinking product and use Qubit fluorescence quantification to determine the concentration of DNA. The quantification concentration should not be less than 5 ng / μL. Store the decrosslinking product at -20℃.

[0077] 5. Construction of third-generation single-molecule libraries For the preparation method of third-generation single-molecule sequencing libraries, please refer to (CN118241319A).

[0078] Samples are fed into a nanopore sequencer, producing 5-6 Gb of data for subsequent bioinformatics analysis.

[0079] 6. Bioinformatics Analysis of Third-Generation Single-Molecular Imaging Data (1) Aligning raw data to the reference genome: The raw data is in fastq format. The sequence is aligned to the human reference genome sequence (hg19 / hg38) using software such as minimap2 / ngmlr, generating the original alignment bam file.

[0080] (2) Preparation of restriction enzyme recognition files: Determine the recognition of restriction enzyme sequences based on the type of restriction endonuclease, such as the DpnII (G / ATC) restriction enzyme in this example. Prepare the location of these restriction sites in the human reference genome (hg19, hg38) using cooler software.

[0081] (3) Enzyme digestion sequence identification: The enzyme digestion sites are identified using HiPro software to obtain the re-alignment results after being cut by the enzyme digestion signal.

[0082] (4) QC quality control information generation: Based on the Juicer software, generate QC quality control files from the results of step (3) and check whether indicators such as fragment size and long range ratio meet the quality control requirements.

[0083] (5) SNP / INDEL variant analysis: Based on the results of step (3), the realigned BAM file was analyzed using DeepVariant software to obtain the SNP / INDEL variant analysis results. (6) Generation of interactive signal files: Pairtools software can generate interactive signal files from the re-alignment results generated in step (3). The results are shown in Tables 2 and 3.

[0084] In this embodiment, the subjects underwent library construction for a second-generation high-throughput chromatin conformation capture library as a control.

[0085] For the experimental protocol of second-generation chromatin conformation capture sequencing, please refer to: Lafontaine DL, Yang L, Dekker J, et al. Hi‐C 3.0: improved protocol for genome‐wide chromosome conformationcapture[J]. Current Protocols, 2021, 1(7): e198. For the bioinformatics analysis workflow of second-generation chromatin conformation capture data, please refer to: Lajoie BR, Dekker J, Kaplan N. The Hitchhiker's guide to Hi-C analysis: practical guidelines[J]. Methods, 2015, 72: 65-75. Table 2 As shown in Table 2, the effective contact interaction (Hi-C Contacts) ratio in the method of this invention is 100%, while it is 76.54% in the second-generation high-throughput chromatin conformation capture method, indicating that the read utilization rate of the method of this invention is higher than that of the second-generation high-throughput chromatin conformation capture method. Secondly, the long-range (>20 kb) chromosome signal interaction ratio in the method of this invention is 35.82%, significantly higher than the 18.71% in the second-generation high-throughput chromatin conformation capture method. The increase in effective reads and long-range signal values ​​is beneficial for the identification of structural variations and the construction of haplotypes.

[0086] Table 3 As shown in Table 3, regardless of the sequencing depth of 3× or 5×, the SV resolution of the method of this invention is higher than that of the second-generation high-throughput chromatin conformation capture method, and the number of SNPs in a 1 Mb chromosome fragment is much higher than that of the second-generation high-throughput chromatin conformation capture method.

[0087] The spatial contact map results of chromosome 9 of sample M-HF-P1 and sample XM-DD1 obtained by the method of this invention are as follows: Figure 1 and Figure 2 As shown, for the repetitive sequences and centromere regions of the chr9 chromosome (red box), the method of this invention has a higher read coverage area compared to the second-generation high-throughput chromatin conformation capture method (one red dot in the figure represents one read). The chromosomal region that the method of this invention cannot cover is approximately between 47 Mb and 65 Mb (18 Mb), while the region that the second-generation high-throughput chromatin conformation capture method cannot cover is approximately between 39 Mb and 72 Mb (33 Mb), which is much larger than the region that the method of this invention cannot cover, indicating that the method of this invention is superior.

[0088] The spatial contact map results of chromosome 16 of sample M-HF-P1 and second-generation chromatin conformation capture sample XM-DD1 obtained by the method of this invention are as follows: Figure 3 and Figure 4As shown, for the 15460000-16330000 segment of the highly homologous chr16 chromosome, the method of this invention also has more read coverage compared to the second-generation high-throughput chromatin conformation capture method (one red dot in the figure represents one read).

[0089] Example 2 A method for constructing and detecting haplotypes in structurally variant-positive samples.

[0090] A karyotype-positive sample (circular chromosome r(21)(p13q22.3)) LCL was selected and processed using the cell processing and library construction methods described in Example 1. The sequencing data alignment, SNP variation analysis, and interaction signal generation procedures followed those in Example 1. Simultaneously, an embryonic biopsy was performed on this sample, and at least five cells were isolated from the trophectoderm of the blastocyst stage embryo. The cellular DNA was amplified and sequenced using the MDA whole-genome amplification kit (REPLI-g Single Cell Kit).

[0091] Bioinformatics haplotype construction process: The process of offline data alignment, SNP mutation analysis, and interaction signal generation is described in Example 1. The haplotype construction process is as follows: (1) Construction of target structural variant chromosome SNP haplotypes: The target chromosome was sequenced using HapCut2 software to obtain two haplotype SNPs (Hap1 and Hap2).

[0092] (2) Extracting chromosome interaction signals carried by structural variations: Based on the results of step (1), the breakpoint of the target structural variation is identified, and the sequence information file (bam file) of the chromosome where the structural variation is located is extracted. Sequence information within 10 Mb upstream and downstream of the breakpoint of the structural variation is extracted. The sequence in chromosome A upstream of the breakpoint is selected, and its associated interaction sequence is located in chromosome B downstream of the breakpoint. Sequences that meet the above conditions are extracted, and the variation of the relevant sequences is re-analyzed. The base with higher depth (greater than 3×) support in the SNP analysis results closest to the breakpoint is extracted as the haplotype base A1 carrying the chromosome. At this time, in the heterozygous SNP information generated in step (7) corresponding to the chromosome position, the other base B1 that is not A1 is taken as the haplotype base B1 of the normal chromosome, and the initial SNP haplotype pair A1 and B1 are obtained. Extending sequentially towards the breakpoint, haplotype pairs A2B2, A3B3...AnBn, etc. that meet the above conditions are searched until the range of 10 Mb upstream of the breakpoint ends. Similarly, within a 10 Mb range downstream of the fracture point, eligible haplotype pairs C1D1, C2D2…CnDn, etc., are extracted. SNP information of structurally variable haplotypes (SVhap1 and SVhap2) within a 10 Mb range upstream and downstream of the fracture point is obtained.

[0093] (3) SNP extension of structural variant haplotypes: If the sites in SVhap1 / SVhap2 that exist in Hap1 / Hap2 in step 7 are retrieved, SVhap1 / SVhap2 can be extended. The extended haplotype range is within the assembly block where Hap1 / Hap2 is located. After all sites are retrieved, the SNP haplotype extension of SVhap1 / SVhap2 is completed.

[0094] (4) Results Output: Based on the above analysis process, the structural variation information of the sample and the SNP haplotype results of the detected structural variations are output, as shown in Tables 4-5 and 5. Figures 5-6 As shown.

[0095] Table 4 Table 5 As shown in Tables 4 and 5, with a data volume of 30G (sequencing depth of 10×), the fragment length of this invention can reach 3.97 kb. When using the haplotype data from hic construction for embryo carrier analysis, regardless of whether the data depth is 10× or 30×, the number of SNP loci effectively genotyped per 1 MB using this method far exceeds the number of loci captured by second-generation high-throughput chromosome cytoplasmic conformation. The more loci, the higher the accuracy of embryo screening and genotyping.

[0096] Figure 5 and Figure 6 The blue line represents the circular chromosome carrying chain of LCL, and the red line represents the normal chain. P01, P02, and P03 are the genotyping results of three embryos from the sample. The genotyping results of the method of this invention are consistent with those of second-generation high-throughput chromatin conformation capture, where P01 and P03 are embryos carrying the circular chromosome, and P02 is an embryo that does not carry it. However, since the effective SNPs that can be used for identification by second-generation high-throughput chromatin conformation capture are limited, there is a certain high proportion of background noise in the genotyping process of P02 (especially the blue dots at the breakpoints q11.2 and q22.3), which may interfere with the accurate interpretation of the results and make it impossible to identify the risk of recombination.

[0097] In summary, the method of this invention can effectively reduce the amount of blood collected, the amount of cells input, and the amount of reagents used, shortening the operation time from 4-5 days to 2 days, and eliminating the need for overnight waiting. Compared with second-generation high-throughput chromatin conformation capture methods, the method of this invention has significant advantages and performance improvements in terms of long-range signal coverage, SNP count, repetitive sequences, centromere coverage, and coverage of highly homologous sequences.

[0098] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for analyzing structural variations based on third-generation single-molecule sequencing, characterized in that, The method includes: fixing and cross-linking the sample to be tested to obtain fixed cells; lysing and permeabilizing the fixed cells; releasing chromatin; using restriction endonucleases to break down the chromatin and perform a ligation reaction to obtain ligation products; decross-linking the ligation products to obtain DNA products; constructing a third-generation single-molecule sequencing library from the DNA products to obtain a sequencing library; performing third-generation single-molecule sequencing on the sequencing library to obtain sequencing data; and performing bioinformatics analysis on the sequencing data to resolve structural variations in the sample, wherein the cell count of the sample to be tested is 0.01 × 10⁻⁶. 6 -100×10 6 indivual.

2. The method for analyzing structural variations based on third-generation single-molecule sequencing according to claim 1, characterized in that, The sample to be tested includes any one or a combination of at least two of the following: blood, blood products, amniotic fluid, chorionic villus tissue, or isolated cells. Preferably, the source of the isolated cells includes any one or a combination of at least two of lymphocytes, stem cells, or bone marrow; Preferably, the sample to be tested is peripheral blood mononuclear cells.

3. The method for analyzing structural variations based on third-generation single-molecule sequencing according to claim 1 or 2, characterized in that, The lysis and permeabilization process includes: lysing fixed cells using lysis buffer and protease inhibitor, followed by permeabilization with sodium dodecyl sulfate and Triton X-100; the total volume of the lysis and permeabilization process is 100-160 μL. Preferably, the restriction endonuclease includes any one or a combination of at least two of DpnII, HinfI, or DdeI; Preferably, the chromatin disruption conditions are 35-38℃ shaking incubation for 0.5-2 h; Preferably, the total volume of the reaction system for disrupting chromatin is 145-150 μL; Preferably, the ligation reaction system includes T4 DNA ligase and ligation buffer; Preferably, the amount of T4 DNA ligase used is 2-5 μL / reaction; Preferably, the ligation buffer contains T4 DNA ligase buffer and polyethylene glycol; Preferably, the temperature of the connection reaction is 20-25°C, and the time of the connection reaction is 0.5-2 h; Preferably, the decrosslinking treatment includes enzymatic hydrolysis using proteinase K and / or streptomycin, followed by shaking incubation at 58-62°C for 0.5-2 h; Preferably, the total volume of the decrosslinking treatment system is 120-200 μL.

4. The method for analyzing structural variations based on third-generation single-molecule sequencing according to any one of claims 1-3, characterized in that, The construction of the third-generation single-molecule sequencing library includes the construction of nanopore sequencing libraries.

5. The method for analyzing structural variations based on third-generation single-molecule sequencing according to any one of claims 1-4, characterized in that, The bioinformatics analysis includes: aligning sequencing data to a reference genome to obtain an alignment file; identifying enzyme restriction sites to obtain re-alignment results; generating QC quality control information; performing SNP / INDEL variant analysis; generating interaction signal files; and identifying structural variants to obtain structural variant breakpoint information. Preferably, the identification of structural variations includes: establishing an interaction signal database of normal samples, removing background noise signals from the samples to be tested, and obtaining accurate structural variation signals.

6. The method for analyzing structural variations based on third-generation single-molecule sequencing according to claim 5, characterized in that, The bioinformatics analysis also includes copy number variation identification and / or SNP haplotype construction of chromosomes with structural variations.

7. The method for analyzing structural variations based on third-generation single-molecule sequencing according to claim 6, characterized in that, The copy number variation identification includes: establishing a copy number reference database for the whole genome of normal samples, calculating the copy number results of the samples to be tested, generating the Log value of the chromosome window, and determining the breakpoint of the copy number difference; Preferably, the construction of the SNP haplotype of the structurally variable chromosome includes the following steps: (1) Sequence assembly of the target chromosome was performed to obtain SNP information of the Hap1 haplotype and Hap2 haplotype of the chromosome; (2) Identify the breakpoint of the target structural variation and extract the sequence information of the chromosome where the structural variation is located; (3) Extract sequences that conform to the structural variation characteristics in the upstream and downstream regions of the breakpoint, analyze their variation, and obtain haplotype bases carrying chromosomes and haplotype bases of normal chromosomes. (4) Extend upstream and downstream of the fracture point to obtain structural variation monomer type SNP information within the upstream and downstream range of the fracture point; (5) Match and extend the monomeric SNP information obtained in step (4) with the monomeric SNP information obtained in step (1) to obtain complete structural variation monomeric SNP information.

8. The application of the method for analyzing structural variations based on third-generation single-molecule sequencing as described in any one of claims 1-7 in the preparation of products for genetic disease screening, preconception screening, tumor research, or structural variation detection.

9. The application according to claim 8, characterized in that, The genetic disease screening includes screening for non-carrier embryos.

10. A detection apparatus for implementing the method for analyzing structural variations based on third-generation single-molecule sequencing as described in any one of claims 1-7, characterized in that, The detection device includes a cell extraction and fixation module, a chromosome conformation capture module, a sequencing library construction module, a sequencing module, a structural variation analysis module, and a haplotype construction module; The cell extraction and fixation module is used to perform the following: extracting cells from a sample and fixing them into a conformation; The chromosome conformation capture module is used to perform actions including: cell lysis and permeation, chromatin fragmentation, ligation, and decrosslinking. The sequencing library construction module is used to perform actions including: constructing a nanopore sequencing library; The sequencing module is used to perform the following operations: sequencing a nanopore sequencing library, aligning the sequencing data to a reference genome to obtain an alignment file; identifying restriction enzyme sites to obtain re-alignment results; generating QC quality control information; performing SNP / INDEL variant analysis; and generating an interaction signal file. The structural variation analysis module is used to perform the following tasks: establishing an interactive signal database of normal samples, removing background noise signals of the test samples, and obtaining accurate structural variation signals. The haplotype construction module is used to perform the following: sequence assembly of the target chromosome, obtaining SNP information of the Hap1 and Hap2 haplotypes of the chromosome, identifying the breakpoint of the target structural variation, extracting the sequence information of the chromosome where the structural variation is located, extracting sequences that conform to the characteristics of the structural variation in the upstream and downstream regions of the breakpoint, analyzing their variation, obtaining the haplotype bases carrying the chromosome and the haplotype bases of the normal chromosome, extending upstream and downstream of the breakpoint to obtain the structural variation haplotype SNP information within the upstream and downstream range of the breakpoint, and matching and extending the obtained haplotype SNP information to obtain complete structural variation haplotype SNP information.

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