Haploid identification method independent of family

Abstract

The invention provides a method for identifying a structure variation linked haplotype, and particularly relates to a method for identifying an embryo structure variation linked haplotype. The method is independent of family information and is particularly suitable for identifying whether an embryo lacking family information carries a chromosomal structural variation in pre-transplantation screening and / or pre-natal screening, thereby implementing variation blocking. The invention also provides a detection product for implementing the method.

Description

Technical Field

[0001] The present invention relates to the field of genetic variation detection, and more specifically, to a method for identifying structural variation linkage haplotypes, especially embryonic structural variation linkage haplotypes, that is independent of family. Background Art

[0002] In the field of biomedicine, chromosome structural variation is a common chromosome genetic defect. The main types of variation include deletion, duplication, insertion, inversion, translocation, etc., and the probability of its occurrence may even reach 10% or higher. Structural variation affects gene function by affecting gene structure, changing gene expression, changing the three-dimensional structure of chromosomes, etc., and is associated with many serious genetic diseases. Although structural variations such as balanced translocation, Robertsonian translocation, inversion, insertion, deletion, etc. are more common in carriers and are usually not accompanied by disease symptoms, carriers are more likely to produce unbalanced gametes, and are more likely to suffer from infertility, habitual miscarriage, or congenital malformations, autism, intellectual development disorders, etc. in their offspring (or offspring of offspring). Therefore, the identification of structural variation and the blocking of its inheritance to offspring (for example, selecting and identifying embryos that do not carry variation for embryo transplantation) are of great significance in the practice of eugenics and artificial assisted reproduction.

[0003] In current practice, it is not difficult to find embryos with abnormal copy numbers through whole genome sequencing, chips and other means. However, embryos with normal copy numbers but carrying mutations such as inversions, translocations, and insertions are relatively difficult to detect through direct testing. An effective identification strategy is to first identify the haplotype of the mutation carrier in both parents, and then determine whether it carries the same structural variation by detecting and comparing the haplotype carrying status of the embryo. Usually, the method of haplotype identification is to more accurately determine the SNPs that are linked to the variation by performing linkage analysis on the carrier's family information, including carrier parents, carrier offspring samples, or embryos with unbalanced copy numbers, and distinguish between the haplotypes where the variation is located and the normal haplotype, so as to select embryos where the normal haplotype that does not carry the variation is located for transplantation. However, this method has an application threshold in reality, especially for genetic families, such as the need to screen out a certain number of embryos with unbalanced copy numbers, and / or sample family members. In addition to the problems of long detection cycle and high cost, many subjects cannot find available family information at all, so they cannot judge the haplotype related to the variation, and cannot achieve accurate and effective variation blocking during reproduction.

[0004] Chromosome conformation capture technologies, such as Hi-C technology, use high-throughput sequencing and bioinformatics analysis methods to obtain the spatial relationship between chromosomes across the entire genome, and have been widely used in genome assembly and structural variation detection. With the help of Hi-C technology and haplotype assembly technology, full-length haplotypes at the chromosome level can be assembled. In theory, variant carriers can be detected by constructing full-length haplotypes of the genome and constructing linked haplotypes, but the cost is very high and it is difficult to apply to clinical testing.

[0005] There are still gaps in the existing technology in the hope of better meeting the testing needs for the purpose of eugenics. Summary of the invention

[0006] The present invention uses Hi-C technology to construct local haplotypes and variant-linked haplotypes based on low-depth sequencing and a single sample, without the need for other embryos or carrier family information, thereby expanding the range of subjects for variant blocking in embryo transplantation.

[0007] In a first aspect, the present invention provides a method for identifying structural variation linkage haplotypes, the method comprising the steps of:

[0008] a1. Obtain biological samples from structural variation carriers and extract genomic DNA;

[0009] a2. The library was constructed and sequenced by chromosome conformation capture technology for the extracted genomic DNA;

[0010] a3. Align the genome sequence measured in step a2 to the human reference genome;

[0011] a4. Determine one or more structural variation breakpoints by combining the sequence aligned to the reference genome in step a3 with the data characteristic signals obtained by sequencing using chromosome conformation capture technology;

[0012] a5. For the sequence aligned to the reference genome in step a3, according to the one or more breakpoint information detected in step a4, detect SNPs upstream and downstream of the one or more breakpoints, and retain heterozygous SNP results;

[0013] a6. For the sequences aligned to the reference genome in step a3, extract the paired-end sequencing information thereof, and screen them according to their genomic positions, with the screening criteria being to retain the paired-end paired sequences whose two ends can be respectively aligned to the upstream and downstream of the breakpoint positions corresponding to the normal chromosomes that have not undergone mutations as the normal chain characteristic sequences, and to retain the paired-end paired sequences whose two ends can be respectively aligned to the upstream and downstream of the breakpoints of the newly generated chromosomes after mutation as the variant chain characteristic sequences, and remove the sequences that do not meet the above criteria;

[0014] a7. Detect SNPs in the sequence retained in step a6, and retain the homozygous SNP results;

[0015] a8. Take the intersection of the SNP sites retained in step a5 and the SNP sites retained in step a7, and compare the effective depths of each site in the intersection in step a5 and step a7, and retain the site whose effective depth in step a5 is greater than the effective depth in step a7 as the haplotype anchor point, wherein at this site, the homozygous base obtained in step a7 is the characteristic base representing the structural variation haplotype, and the other of the heterozygous bases obtained in step a5 is the characteristic base representing the normal chromosome;

[0016] a9. Assemble and construct local linkage SNP blocks for the sequences aligned to the reference genome in step a3;

[0017] a10. In the local linked SNP block obtained in step a9, search for the position of the haplotype anchor point obtained in step a8. For the block containing the anchor point SNP, use the base detected in step 8 as a reference, and use the SNP allele linked to the base as the linked SNP allele representing the structural variation haplotype, and the other as the linked SNP allele representing the normal haplotype.

[0018] In some embodiments, the chromosome conformation capture technology in step a2 is Hi-C technology.

[0019] In some embodiments, the reference genome in step a3 is hg19, hg38, T2T, etc.

[0020] In some embodiments, the alignment is performed in step a3 by genome alignment tool software. In some preferred embodiments, the software is BWA, samtools, Bowtie2, HiC-Pro and / or hisat2.

[0021] In some embodiments, the breakpoints are detected in step a4 by Hi-C data analysis software. In some preferred embodiments, the software is juicer.

[0022] In some embodiments, the one or more structural variation breakpoints detected in step a4 include normal chain breakpoints and / or variant chain breakpoints. In some embodiments, the variant chain breakpoints include variant position breakpoints and / or variant sequence breakpoints.

[0023] In some embodiments, the software used to detect SNP in step a5 is GATK or freebayes.

[0024] In some embodiments, the range upstream and downstream of the breakpoint in step a5 is preferably 10 Mbp upstream and downstream of a balanced translocation breakpoint, 10 Mbp downstream of a Roche ectopic breakpoint, 5 Mbp upstream and downstream of an inversion breakpoint, 10 Mbp upstream and downstream of an insertion variation breakpoint, and 10 Mbp upstream and downstream of a deletion variation breakpoint.

[0025] In some embodiments, the range upstream and downstream of the breakpoint in step a6 is 10 Mbp upstream and downstream of the balanced translocation breakpoint, 10 Mbp downstream of the Roche ectopic breakpoint, 5 Mbp upstream and downstream of the inversion breakpoint, 10 Mbp upstream and downstream of the insertion variation breakpoint, and 10 Mbp upstream and downstream of the deletion variation breakpoint.

[0026] In some embodiments, in step a9, haploid assembly analysis software is used to assemble and construct local linkage SNP blocks. In some preferred embodiments, the software is HapCUT or HapCUT2, more preferably HapCUT2.

[0027] In some embodiments, the structural variation is selected from: structural rearrangement and copy number abnormality (CNV), preferably structural rearrangement. In some specific embodiments, the structural rearrangement structural variation is selected from translocation (balanced translocation, unbalanced translocation, reciprocal translocation, non-reciprocal translocation, Robertsonian translocation and complex translocation), inversion (intra-arm inversion and inter-arm inversion), and insertion (forward insertion and inversion insertion). In some embodiments, the copy number abnormality structural variation is selected from deletion and duplication. In some embodiments, the structural variation can be a chromosome structural change with sequence breakpoints such as isochromosomes and ring chromosomes.

[0028] In some embodiments, the sequencing is a low-depth double-end sequencing. In some embodiments, the sequencing depth is not less than 1X, not less than 2X, not less than 3X, not less than 4X, or not less than 5X. In some embodiments, the sequencing depth is not higher than 15X, not higher than 14X, not higher than 13X, not higher than 12X, not higher than 11X, not higher than 10X, not higher than 9X, not higher than 8X, not higher than 7X, not higher than 6X, or not higher than 5X. In some embodiments, the sequencing depth is not less than 3X, about 30M reads, wherein preferably, Pair-ended sequencing is used. In some specific embodiments, the sequencing data amount is about 40M reads. In some specific embodiments, the sequencing data amount is about 30M reads to about 40M reads.

[0029] In a second aspect, the present invention further provides a method for identifying the linked haplotype status of a structural variation in an embryo, the method comprising the steps of:

[0030] b1. Sampling: obtaining embryo biopsy cells and peripheral blood samples from both parents;

[0031] b2. using the method for identifying structural variation-linked haplotypes of the first aspect of the present invention, identifying structural variation-linked haplotypes of the structural variation carriers in the parents, and obtaining one or more breakpoint information related to the variation and two allele information of the linked SNP allele representing the structural variation haplotype and the linked SNP allele representing the normal haplotype;

[0032] b3. Extract DNA from embryo biopsy cells and peripheral blood samples of the non-carrier parent, amplify, build a library and then sequence;

[0033] b4. Align the genome sequences of the two samples measured in step b3 to the same human reference genome;

[0034] b5. For the sequence aligned to the reference genome in step b4, detect SNPs upstream and downstream of the breakpoints according to the mutation-related breakpoint information of the carrier;

[0035] b6. Intersecting the variant haplotype-linked SNP result obtained in step a10 of the method of the first aspect of the present invention with the SNP result obtained in step b5 according to their positions on the genome to infer which allele of the carrier is inherited by the embryo at that position, thereby independently determining for each SNP site of the embryo whether its genotype is consistent with the variant haplotype-linked SNP allele or consistent with the normal haplotype SNP allele;

[0036] b7. Count the number of SNP sites determined in step b6 upstream and downstream of the structural variation-related breakpoints. If the number of SNP sites consistent with the variant haplotype-linked SNP allele is greater than the number of sites consistent with the normal haplotype, then the embryo is considered to carry the structural variation haplotype obtained in step a10 of the first aspect, that is, it is presumed to carry the variation. Otherwise, it is presumed not to carry the variation.

[0037] In some embodiments, the biopsied cells of the embryo in step b1 are single or multiple cells from the blastocyst trophoblast. In some preferred embodiments, the blastocyst is a healthy and high-quality blastocyst cultured to day 5-8 after egg retrieval.

[0038] In some embodiments, the sequencing in step b3 is performed by NGS sequencing after single-cell amplification and library construction using the MALBAC method.

[0039] In some embodiments, the range upstream and downstream of the breakpoint in step b5 is preferably 10 Mbp upstream and downstream of a balanced translocation breakpoint, 10 Mbp downstream of a Roche ectopic breakpoint, 5 Mbp upstream and downstream of an inversion breakpoint, 10 Mbp upstream and downstream of an insertion variation breakpoint, and 10 Mbp upstream and downstream of a deletion variation breakpoint.

[0040] In some embodiments, the range upstream and downstream of the breakpoint in step b6 is preferably 10 Mbp upstream and downstream of a balanced translocation breakpoint, 10 Mbp downstream of a Roche ectopic breakpoint, 5 Mbp upstream and downstream of an inversion breakpoint, 10 Mbp upstream and downstream of an insertion variation breakpoint, and 10 Mbp upstream and downstream of a deletion variation breakpoint.

[0041] In some embodiments, the inference method in step b6 is: when the non-carrier (i.e., the carrier spouse) carries a homozygous SNP, no matter whether the embryo is homozygous or heterozygous at this site, there must be an allele that is a homozygous base from the non-carrier, then the other allele in the embryo can be identified as an allele from the carrier; and when the embryo carries a homozygous SNP, even if the spouse's base is uncertain, it can be considered that one of the alleles in the homozygous SNP is inherited from the carrier.

[0042] In a third aspect, the present invention provides a detection product for implementing the aforementioned method of the present invention, which comprises one or more of the following modules:

[0043] (1) gDNA extraction module: used to extract genomic DNA from a sample (e.g., a cell);

[0044] (2) Chromosome conformation capture module: used to fix the chromosome conformation in the sample, that is, to fix the interactions between spatially contacting or adjacent DNA fragments and / or with proteins, for example, by covalent cross-linking, so that they can be detected in subsequent steps;

[0045] (3) Sequencing library construction module: used to generate a whole genome DNA sequencing library;

[0046] (4) Sequencing module: used for low-depth double-end sequencing of the sequencing library;

[0047] (5) Alignment module: used to align the whole genome sequencing results generated by the sequencing module to the reference genome;

[0048] (6) Structural variation assessment module: used to assess the chromosome structural variation of sample genomic DNA from sequencing data;

[0049] (7) SNP detection module: used to detect SNP sites and their genotypes from sequencing data;

[0050] (8) Typing module: used to compare and screen related SNP sites and identify haplotype-related linked SNP alleles.

[0051] In some preferred embodiments, the chromosome conformation capture module and / or sequencing library construction module is based on Hi-C technology.

[0052] In some preferred embodiments, the detection product is a detection system. In some preferred embodiments, the detection system comprises a computer and / or storable software. In some preferred embodiments, the computer is connected to a sequencer.

[0053] In a fourth aspect, the present invention provides the use of the identification method / detection method according to the present invention, or the detection product according to the present invention for pre-embryo implantation screening and / or prenatal screening, or for the preparation of a product for pre-embryo implantation screening and / or prenatal screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Shown is a spatial contact matrix obtained after genomic HiC sequencing of a sample from a subject carrying a balanced chromosomal translocation.

[0055] Figure 2 Figure 2a and Figure 2b ) shows the results of genotyping of haplotype-related linked SNP loci for both parents (one of them carries a balanced translocation), and the results of genotyping of embryonic cells of their candidate offspring, where Figure 2a and Figure 2b A portion of representative SNP sites are shown.

[0056] Figure 3 Figure 3a and Figure 3b ) shows the results of genotyping of haplotype-related linked SNP loci for both parents (one of whom carries Roberta's translocation), and the results of genotyping of embryonic cells of their candidate offspring, where Figure 3a and Figure 3b A portion of representative SNP sites are shown. Since the short arm of the chromosome is lost after translocation, the SNP sites are marked as the distance downstream of the breakpoint.

[0057] Figure 4 Shown is a spatial contact matrix obtained after genomic HiC sequencing of a sample from a subject carrying a chromosomal inversion.

[0058] Figure 5 The results of genotyping of haplotype-related linked SNP loci for both parents (one of whom carries the inversion) and the results of genotyping of embryonic cells of their candidate offspring are displayed.

[0059] Figure 6 Shown is the spatial contact matrix obtained after genomic HiC sequencing of a sample from a subject carrying a chromosomal insertion variant.

[0060] Figure 7The results of haplotype-related linked SNP genotype identification for both parents (one of them carries the insertion variant) and the results of genotype identification contained in the embryonic cells of their candidate offspring are shown. The SNP sites are marked as the distance upstream and downstream of the insertion site.

[0061] Figure 8 Schematic diagram showing copy number balanced insertion variants and their corresponding deletion variants, as well as the identification of the normal chain and variant chain in the two types of variants.

[0062] Fig. 9 Shown is the spatial contact matrix obtained after genomic HiC sequencing of a sample from a subject carrying a chromosomal insertion variant.

[0063] Fig.10 The results of haplotype-related linked SNP genotype identification for both parents (one of them carries the insertion variant) and the results of genotype identification contained in the embryonic cells of their candidate offspring are shown. The SNP sites are marked as the distance upstream and downstream of the insertion site. DETAILED DESCRIPTION OF THE INVENTION

[0065] definition

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. See, for example, Lackie, DICTIONARY OF CELL AND MOLECULARBIOLOGY, Elsevier (4th edition 2007); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, New York 1989).

[0067] Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of the present invention. The definitions provided herein are intended to aid in the understanding of certain terms frequently used herein and do not limit the scope of the present invention.

[0068] In this article, the term "comprise" and its various variations such as "include" and "contain" are used to indicate that adding other steps or elements is optional and non-exclusive when they are located before the narration steps or elements. In this article, it should be understood that the term "comprise" or its various variations also encompass the scheme consisting of the steps or elements described.

[0069] In this article, when referring to a specific value, the term "about" refers to the usual error range of the value known to those skilled in the art. In this article, it should be understood that the expression covers the reference to the specific value itself. Thus, for example, when referring to "about X", a specific reference to the specific value X itself is also covered.

[0070] In this article, the term "chromosome" is used interchangeably with "chromatin" and refers to a complex of chromosomes that contain all or part of the genome of a cell. The genome of a cell is generally characterized by its karyotype, which is a collection of all chromosomes that contain the genome of a cell. The genome of a cell may include one or more chromosomes. In humans, each chromosome has a short arm (called "p") and a long arm (called "q"). Each chromosome arm is divided into regions or bands that can be seen in conventional karyotype analysis using a microscope. Chromosome bands are labeled as p1, p2, p3, etc., and are counted from the centromere toward the telomere. The higher resolution sub-bands in the band are sometimes also used to identify regions in the chromosome. The sub-bands are also numbered from the centromere toward the telomere. Information about chromosome bands and chromosome nomenclature can be found in Strachan, T. and Read, AP 1999. Human Molecular Genetics, 2nd Edition New York: Pages 37-39 of John Wiley & Sons.

[0071] In this article, the term "allele" or "allele" generally means a diverse candidate DNA sequence (alternative form) on the identical entity locus in a DNA segment (e.g., on homologous chromosomes). In diploid cells or organisms, two alleles (identical (homozygous) or different (heterozygous)) of a particular gene occupy a corresponding locus on a pair of homologous chromosomes. "Difference" between different alleles can correspond to a single nucleotide difference (e.g., SNP) in the sequence on the entity locus, or can correspond to multiple groups of differences, e.g., multiple groups of differences linked, such as haploid (type). Conceptually, allele can mean a DNA sequence dna that differs between multiple identical entity loci found on homologous chromosomes in a single cell or an individual organism, or can mean or differ on the identical entity loci in multiple cells or organisms (allelic variants), if not otherwise specified, mainly relate to the former in this article.

[0072] Herein, the term "chromosome structural variation", also referred to as "chromatin structural variation", refers to the structural difference of the chromosomes of an individual organism relative to the chromosomes of other individuals within the same species. The structural difference can be a structural rearrangement of the chromosome and a copy number variation; and covers chromosome structural changes of various sizes, for example, 1-5M, 5-10M, more than 10M, or even a large part of an individual chromosome, such as half, one-third or three-quarters of the structural changes. Non-limiting examples of types of chromosome structural variation include translocation, reciprocal translocation, non-reciprocal translocation, balanced translocation, unbalanced translocation, complex translocation, inversion, insertion, deletion, duplication, repeat amplification, and a combination of two or more of the same or different types of variation.

[0073] As used herein, the term "chromosome structural rearrangement" or "structural rearrangement" refers to a change in the order and position of a DNA sequence on a chromosome. A structural rearrangement may be a change in position of a chromosomal DNA sequence, such as a translocation; a change in direction, such as an inversion; or a change in type, such as the insertion of a non-homologous chromosome fragment. A structural rearrangement may be an interchromosomal rearrangement; or an intrachromosomal rearrangement.

[0074] In this article, the term "translocation" refers to a chromosome breakage and its whole or part is reconnected to different chromosome segments, for example, between non-homologous chromosomes and / or between two or more positions on the same chromosome. Translocation usually causes the abnormal proximity of two chromosome regions that are not adjacent to each other. Depending on the breakage and reconnection position of the translocation, translocation may have no effect on gene expression, or may affect the expression of a single gene, or may affect the expression of multiple genes. The type of translocation includes, but is not limited to, reciprocal translocation, non-reciprocal translocation, Robertsonian translocation, balanced translocation, unbalanced translocation.

[0075] As used herein, the term "balanced translocation" refers to a translocation form in which no increase or decrease of genetic material occurs during the exchange of genetic material.

[0076] As used herein, the term "unbalanced translocation" refers to a form of translocation in which a loss of genetic material occurs during the exchange of genetic material.

[0077] As used herein, the term "reciprocal translocation" refers to the exchange of segments between two chromosomes. Balanced reciprocal translocation carriers are often phenotypically normal, but may cause unbalanced chromosomal translocations in gametes, leading to infertility, miscarriage, or phenotypic abnormalities in offspring.

[0078] As used herein, the term "nonreciprocal translocation" refers to the unidirectional transfer of genetic material from one chromosome to another nonhomologous chromosome.

[0079] In this article, the term "Robertsonian translocation" or "Robertsonian translocation" refers to a translocation caused by the breakage of two chromosomes with proximal centromeres at or near the centromere, in which the long arms of the two chromosomes are reconnected to form a translocated chromosome, while the two short arms form a very small chromosome and are often lost. Robertsonian translocation usually occurs between human chromosomes 13, 14, 15, 21, and 22, with an incidence of about 1.23 / 1000 in the population and about 2-3% in the infertile population. Robertsonian translocation carriers have only 45 chromosomes, but often have a normal phenotype.

[0080] As used herein, the term "complex translocation" refers to a translocation involving more than two chromosomes and more than two breakpoints. Complex translocations can be balanced or unbalanced.

[0081] In this article, the term "inversion" refers to a chromosome structural rearrangement that changes the direction of the DNA sequence in the chromosome. In this process, the broken fragments produced by the two breaks of the same chromosome are reconnected after being reversed 180 degrees. Inversion can be an intra-arm inversion and an inter-arm inversion. An intra-arm inversion refers to an inversion that occurs on the same arm (e.g., long arm or short arm) of a chromosome. An inter-arm inversion refers to an inversion of a chromosome fragment that contains a centromere region.

[0082] In this article, the term "insertion" refers to the process in which a DNA fragment on a chromosome moves from its original position to another position on the same or different chromosome. Insertion mutation usually does not cause an increase or decrease in genetic material, so the genetic material of the insertion mutation is usually balanced.

[0083] As used herein, the term "breakpoint" refers to a site or region of a chromosome where a chromosome break occurs during a structural change such as a translocation or an inversion.

[0084] As used herein, the term "copy number variation" (CNV) refers to a change in the number of copies of a chromosomal DNA sequence. A CNV can be a copy number variation of an entire chromosome or a chromosome segment, such as an increase or decrease in the number of copies.

[0085] As used herein, the term "read" or "read(s)" refers to a short nucleotide sequence generated by any sequencing process described herein or known in the art. In double-end sequencing, a pair of reads (also referred to as a read pair) with a physical relationship is generated from both ends of the sequenced nucleic acid. Pair-end reads obtained by pair-ended sequencing can be referred to as PEreads.

[0086] In this article, the term "read length" refers to the nucleotide length of the read. The length of the sequence read is usually associated with a specific sequencing technology. For example, next-generation sequencing (NGS) can provide sequence reads ranging from tens to hundreds of base pairs (bp) in length.

[0087] In this article, the term "haplotype (Haplotype, Haploid genotype)" or "haplotype", "haploid type", "unit type" refers to a combination of two or more genetic variation sites coexisting on a single chromosome. Usually, the combination is completely inherited from a group of alleles from one parent of both parents. In a haplotype, the probability of the specific allele type of the two or more variation sites appearing at the same time is higher than the frequency of random occurrence. This relationship is called linkage disequilibrium, and its principle is based on the non-random association between alleles, that is, alleles on different loci are not inherited independently, but show a certain degree of linkage. The linkage disequilibrium on the same chromosome is a haplotype.

[0088] In this article, "sequencing data volume" is given in units of reads. For paired-end sequencing, a pair of PE reads is counted as only one piece of data for the purpose of calculating the volume. For example, when 20 million sequencing data are obtained through pair-ended sequencing, the sequencing data volume can be expressed as 20M.

[0089] In this article, the term "sequencing depth" refers to the ratio of the total number of bases sequenced to the size of the genome being measured. Sequencing depth can be calculated using the following formula:

[0090] Sequencing depth = read length × total number of reads / length of the measured genome sequence.

[0091] In this article, unless otherwise specified in the context, the terms "detecting SNPs", "SNP detection" and "SNP calling" have the same meanings known in the art and can be used interchangeably, namely: identifying single nucleotide polymorphisms (SNPs) at various positions of the reference genome in the sequencing result data based on the sequencing information, and the output results include the genomic coordinates of the SNP, the homozygous / heterozygous genotype and the corresponding base information.

[0092] In this article, the term "joined DNA fragment" or "joined fragment" related to chromosome conformation capture refers to a joined DNA fragment containing a joining point inside formed by end joining of adjacent DNA digested fragments in a covalently cross-linked DNA / protein complex after enzyme digestion during chromosome conformation capture. Accordingly, in this article, the term "unjoined DNA fragment" or "unjoined fragment" refers to an enzyme digested DNA fragment without an internal joining point produced when the end of the enzyme digested DNA fragment is not joined. In conventional chromosome conformation capture technology, it is generally believed that it is necessary to distinguish and separate joined DNA fragments and unjoined fragments, and this distinction and separation is typically performed by biotin-labeling the ends of digested DNA and removing the terminal biotin label of the unjoined fragment after connection. However, the inventors surprisingly found that, as shown in the examples of the present application, according to some embodiments of the method of the present invention, when the enzyme digestion product is used to construct a sequencing library, it is not necessary to distinguish and remove such unjoined fragments from the joined fragments, and good sequencing library quality can still be obtained. It should be understood herein that "joined fragments" also encompass derivative fragments generated from the fragments in library construction (e.g., small fragments generated by mechanical or enzymatic action), as long as the derivative fragments retain the internal joining sites characteristic of the joined fragments from which they are derived. Similarly, "unjoined fragments" also encompass derivative fragments generated from the fragments in library construction (e.g., small fragments generated by mechanical or enzymatic action in library construction), as long as the derivative fragments retain at least one enzyme digestion end characteristic of the unjoined fragments from which they are derived, or enzyme digestion ends modified, labeled and / or repaired accordingly in the case of terminal modification, labeling and / or terminal repair. Therefore, as is clear to those skilled in the art, the term "unjoined fragment" encompasses the terminal small DNA fragments and their derivatives generated by interrupting the fragments, which retain the enzyme digestion ends, but does not encompass internal small DNA fragments or their derivatives generated by interrupting the fragments.

[0093] In this article, chromosome conformation capture technology, such as Hi-C technology, refers to a technology based on chromosome conformation capture (chromatin conformation capture) and sequencing (such as NGS), capturing the interaction information between chromosome DNA fragments in cells. High-throughput chromosome conformation capture technology (such as Hi-C technology) digests and reconnects chromosome fragments close in space, and performs high-throughput sequencing on them, which can determine the spatial interaction between different sites of chromosomes. This technology typically includes two stages, that is, the sequencing library construction of the first stage and the sequencing and bioinformatics analysis of the second stage. In the first stage, sequencing library construction generally involves: by chromosome conformation capture method, chromosome DNA / protein is covalently cross-linked, nuclease digested and reconnected to capture chromosome DNA fragments that interact in space, and generate sequencing library. In the second stage, the library is sequenced to obtain chromosome conformation capture sequencing data, which is also referred to as sequencing data in this article.

[0094] In this article, the spatial contact matrix, sometimes referred to as a genome interaction matrix or Hi-C contact matrix in this article, refers to a two-dimensional matrix generated based on chromosome conformation capture sequencing data to represent the interaction between DNA sequences in the genome, which can generally be used to study the spatial organization of chromosome three-dimensional structure and genome. The rows and columns of the matrix represent the chromosome position interval on the chromosome coordinates; the matrix elements are the number of paired end sequence fragments falling into the corresponding row and column interaction intervals (also referred to as "contact positions" in this article), also referred to as contact intensity / density or interaction frequency (interaction frequency, IF). The spatial contact matrix can be a numerical matrix or can be presented in the form of a visual spatial contact matrix diagram (contact map). In the spatial contact matrix diagram, the change in contact intensity can be reflected by the gradual change of color, thereby presenting color blocks of different color intensities on matrix positions with different contact intensities. In some embodiments, the spatial contact matrix is ​​implemented or generated by computer software. In some embodiments, the computer software is juicer (see Durand NC et al. Juicer Provides a One-Click System for Analyzing Loop-Resolution Hi-C Experiments. Cell Syst. 2016 Jul; 3(1): 95-8.).

[0095] As used herein, the term "module" refers to reagents, sets of reagents, components, sets of components, and / or devices or systems that can be used to implement the function of the module. It should be understood that the composition of the module is not subject to the constraints of a specific physical form, as long as it can achieve the desired function. Depending on the intended function, the module can be a combination of reagents and / or devices that implement the function; or it can be a software object or routine (e.g., as an independent thread) that is executed on a single computing system (e.g., a computer program, a tablet computer (PAD), one or more processors). For example, the chromosome conformation capture module according to the present invention can be composed of reagents or sets of reagents for implementing covalent cross-linking, enzyme digestion and reconnection of chromosome DNA / protein, and optionally a device that can be used to ensure that this process is implemented under specific conditions. For another example, the SNP detection module of the present invention can be a program stored on a computer-readable medium, which contains a computer program logic or code portion for implementing the SNP detection.

[0096] Method of the present invention

[0097] The present invention improves the existing method mainly from three aspects:

[0098] 1. This method can simultaneously detect precise structural variation breakpoints in the step of constructing variant carrier haplotypes, without relying on karyotype or CNV indirect inference to obtain breakpoint location information. This simplifies the analysis process, allowing variation detection and genetic blocking to be completed in one test. On the other hand, the breakpoints obtained by this scheme are highly accurate, reducing the possibility of being unable to judge the results due to recombination near the breakpoints.

[0099] 2. This method does not require family information to infer haplotypes, which expands the scope of application and reduces the probability of detection failure.

[0100] 3. In the conventional variant blocking process, when there is no family information, third-generation sequencing and other technologies are usually used to complete the haplotype construction. In comparison, this method greatly reduces the detection cost and can obtain more effective SNP sites than third-generation sequencing.

[0101] In one embodiment of the present invention, a method for identifying structural variation-linked haplotypes is provided, the method comprising one or more of the following steps:

[0102] a1. Obtain biological samples from structural variation carriers and extract genomic DNA;

[0103] a2. The library was constructed and sequenced by chromosome conformation capture technology for the extracted genomic DNA;

[0104] a3. Align the genome sequence measured in step a2 to the human reference genome;

[0105] a4. Determine one or more structural variation breakpoints by combining the sequence aligned to the reference genome in step a3 with the data characteristic signals obtained by sequencing using chromosome conformation capture technology;

[0106] a5. For the sequence aligned to the reference genome in step a3, according to the one or more breakpoint information detected in step a4, detect SNPs upstream and downstream of the one or more breakpoints, and retain heterozygous SNP results;

[0107] a6. For the sequences aligned to the reference genome in step a3, extract the paired-end sequencing information thereof, and screen them according to their genomic positions, with the screening criteria being to retain the paired-end paired sequences whose two ends can be respectively aligned to the upstream and downstream of the breakpoint positions corresponding to the normal chromosomes that have not undergone mutations as the normal chain characteristic sequences, and to retain the paired-end paired sequences whose two ends can be respectively aligned to the upstream and downstream of the breakpoints of the newly generated chromosomes after mutation as the variant chain characteristic sequences, and remove the sequences that do not meet the above criteria;

[0108] a7. Detect SNPs in the sequence retained in step a6, and retain the homozygous SNP results;

[0109] a8. Take the intersection of the SNP sites retained in step a5 and the SNP sites retained in step a7, and compare the effective depths of each site in the intersection in steps a5 and a7, and retain the site whose effective depth in step a5 is greater than that in step a7 as a haplotype anchor point, wherein at this site, the homozygous base obtained in step a7 is a characteristic base representing the structural variation haplotype, and the other of the heterozygous bases obtained in step a5 is a characteristic base representing a normal chromosome;

[0110] a9. Assemble and construct local linkage SNP blocks for the sequences aligned to the reference genome in step a3;

[0111] a10. In the local linked SNP block obtained in step a9, search for the position of the haplotype anchor point obtained in step a8. For the block containing the anchor point SNP, use the base detected in step 8 as a reference, and use the SNP allele linked to the base as the linked SNP allele representing the structural variation haplotype, and the other as the linked SNP allele representing the normal haplotype.

[0112] In some embodiments, the chromosome conformation capture technology in step a2 is Hi-C technology.

[0113] In some embodiments, the reference genome in step a3 is hg19, hg38, T2T, etc.

[0114] In some embodiments, the alignment is performed in step a3 by genome alignment tool software. In some preferred embodiments, the software is BWA, samtools, Bowtie2, HiC-Pro and / or hisat2.

[0115] In some embodiments, the breakpoints are detected in step a4 by Hi-C data analysis software. In some preferred embodiments, the software is juicer.

[0116] In some embodiments, the one or more structural variation breakpoints detected in step a4 include normal chain breakpoints and / or variant chain breakpoints. In some embodiments, the variant chain breakpoints include variant position breakpoints and / or variant sequence breakpoints.

[0117] In some embodiments, the software used to detect SNP in step a5 is GATK or freebayes.

[0118] In some embodiments, the range upstream and downstream of the breakpoint in step a5 is preferably 10 Mbp upstream and downstream of a balanced translocation breakpoint, 10 Mbp downstream of a Roche ectopic breakpoint, 5 Mbp upstream and downstream of an inversion breakpoint, 10 Mbp upstream and downstream of an insertion variant breakpoint, and 10 Mbp upstream and downstream of a deletion variant breakpoint.

[0119] In some embodiments, the range upstream and downstream of the breakpoint in step a6 is 10 Mbp upstream and downstream of the balanced translocation breakpoint, 10 Mbp downstream of the Roche ectopic breakpoint, 5 Mbp upstream and downstream of the inversion breakpoint, 10 Mbp upstream and downstream of the insertion variant breakpoint, and 10 Mbp upstream and downstream of the deletion variant breakpoint.

[0120] In some embodiments, in step a9, haploid assembly analysis software is used to assemble and construct local linkage SNP blocks. In some preferred embodiments, the software is HapCUT or HapCUT2, more preferably HapCUT2.

[0121] In some embodiments, the structural variation is selected from: structural rearrangement and copy number abnormality (CNV), preferably structural rearrangement. In some specific embodiments, the structural rearrangement structural variation is selected from translocation (balanced translocation, unbalanced translocation, reciprocal translocation, non-reciprocal translocation, Robertsonian translocation and complex translocation), inversion (intra-arm inversion and inter-arm inversion), and insertion. In some embodiments, the copy number abnormality structural variation is selected from deletion and duplication. In some embodiments, the structural variation can be a chromosome structural change with sequence breakpoints such as isochromosomes and ring chromosomes.

[0122] In some embodiments, the sequencing is a low-depth double-end sequencing. In some embodiments, the sequencing depth is not less than 1X, not less than 2X, not less than 3X, not less than 4X, or not less than 5X. In some embodiments, the sequencing depth is not higher than 15X, not higher than 14X, not higher than 13X, not higher than 12X, not higher than 11X, not higher than 10X, or not higher than 9X. In some embodiments, the sequencing depth is not less than 3X, about 30M reads, wherein preferably, Pair-ended sequencing is used. In some specific embodiments, the sequencing data amount is about 40M reads.

[0123] Sequencing

[0124] sample

[0125] The method according to the present invention can be applied to any sample containing nucleated cells. Such samples can be obtained from suitable subjects, such as eukaryotic individuals and mammalian individuals. In some embodiments, the subject is a mammal, preferably a human. In some embodiments, the subject is a non-diseased or diseased individual. In some embodiments, the subject is a human individual to be subjected to prenatal screening.

[0126] The sample can be a sample directly isolated or obtained from a single or multiple subjects or their parts. Examples of samples include, but are not limited to, body fluids or tissues from mammalian individuals, including but not limited to blood or blood products (e.g., serum, plasma, platelets, buffy coat, etc.), cord blood, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., lung, stomach, peritoneum, catheter, ear, arthroscopy), biopsy samples, isolated cells (lymphocytes, placental cells, stem cells, bone marrow-derived cells, embryonic cells such as blastocyst trophoblast cells), or combinations thereof.

[0127] In some embodiments or steps, the sample is a mammalian nucleated cell, body fluid or tissue sample. In some preferred embodiments or steps, the sample is an isolated peripheral blood cell sample. In some preferred embodiments or steps, the sample is an isolated peripheral blood mononuclear cell (PBMC) sample. In some preferred embodiments or steps, the sample is a blastocyst trophoblast cell sample. In some embodiments or steps, the sample contains 0.1x10 6 Up to 10x10 6 nucleated cells, for example, 0.5x10 6 pcs, 1x10 6 pcs, 2x10 6 pcs, 3x10 6 pcs, 4x10 6 pcs, 5x10 6pcs, 6x10 6 pcs, 7x10 6 pcs, 8x10 6 pcs, 9x10 6 10x10 6 In some embodiments or steps, the sample comprises 1x10 6 Up to 5x10 6 nucleated cells, e.g., 1x10 6 Up to 3x10 6 nucleated cells, e.g., no more than 2x10 6 nucleated cells. In some embodiments or steps, the sample contains a very small number of cells, for example, no more than 100 cells, no more than 90 cells, no more than 80 cells, no more than 70 cells, no more than 60 cells, no more than 50 cells, no more than 40 cells, no more than 30 cells, no more than 20 cells, no more than 10 cells, no more than 9 cells, no more than 8 cells, no more than 7 cells, no more than 6 cells, no more than 5 cells, no more than 4 cells, no more than 3 cells, no more than 2 cells, no more than 1 cell. Therefore, in some embodiments or steps, the present invention uses single-cell sequencing technology / platform.

[0128] In some embodiments, the genomic DNA extracted from the sample is purified, for example, using magnetic beads for purification. In some specific embodiments, the purification is performed before sequencing.

[0129] Library construction

[0130] The sequencing library can be established using methods known in the art.

[0131] In order to generate the fragment size suitable for sequencing library construction, a variety of mechanical, chemical and / or enzymatic methods can be used to fragment or shear the DNA in the enzyme digestion product to the desired length. Fragmentation can be performed by ultrasonic treatment, or by briefly exposing to a mixture of DNA enzymes or one or more restriction enzymes, or transposases or nickases. Many enzyme cutting and interruption kits are commercially available, such as from Takara. After fragmentation, the size screening of the fragments can be performed using methods known in the art; and the addition of sequencing adapters.

[0132] In some embodiments, therefore, the method of the present invention comprises: fragmenting the DNA in the enzyme digestion product according to the present invention and screening 150bp-600bp (preferably 300-500bp) fragments for library construction. Preferably, the fragment screening uses two-step magnetic bead addition to recover fragments of appropriate size. The screening of fragments can be performed by adjusting the magnetic bead ratio. In one embodiment, the DNA fragment screening is implemented by adding magnetic beads to the fragmented DNA product in two steps at a 0.6x magnetic bead ratio and a 0.4x magnetic bead ratio.

[0133] Sequencing

[0134] The sequencing can be performed by a double-end sequencing method known in the art. In double-end sequencing, typically, the two ends of the nucleic acid fragment are sequenced with a suitable read length, and the read length is sufficient to map each read (e.g., the read at the two ends of the fragment template) to the reference genome. For example, the read length of the double-end read can be about 10 continuous nucleotides to about 500 continuous nucleotides, about 10 continuous nucleotides to about 400 continuous nucleotides, about 10 continuous nucleotides to about 300 continuous nucleotides, about 50 continuous nucleotides to about 200 continuous nucleotides, about 100 continuous nucleotides to about 200 continuous nucleotides, or about 100 continuous nucleotides to about 150 continuous nucleotides.

[0135] In some embodiments, a paired-end sequencing method is used. In some embodiments, the sequencing depth is less than 6x, for example, the sequencing depth is about 0.1x, 0.3x, 0.5x, 0.8x, 1x, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, and 5x.

[0136] In some preferred embodiments, the sequencing method is MALBAC sequencing, see Chenghang Zong et al., Genome-Wide Detection of Single-Nucleotide and Copy-Number Variations of a Single Human Cell. Science 338, 1622-1626 (2012).

[0137] Detection of chromosome structural variation

[0138] In this article, chromosome structural variations (structural variations) refer to structural changes in the genome. These changes may include large-scale rearrangements, inversions, insertions, translocations, duplications or deletions, as well as small-scale structural variation events, such as copy number abnormalities (CNVs, such as duplications or deletions) of 1M-50M. Since the primary structure is the basis of the spatial structure, the abnormality of the primary structure of DNA often brings about changes in the 3D structure of the chromosome. Therefore, these chromosome structural variations can be identified using chromosome capture technology.

[0139] In chromosome conformation capture sequencing data (e.g., Hi-C sequencing data), the interaction frequency between chromosome regions varies with the genomic distance in a power law distribution. When structural variation causes changes in the genomic structure, two chromosome regions that were originally far apart are close to each other in actual position, causing their interaction frequency to increase, reaching the level of interaction between close-range chromosome regions, thus significantly deviating from the situation in normal chromosomes. For example, the loss of a genomic fragment will cause the two ends of the deleted fragment to be close in spatial position, and thus the interaction frequency of the chromosome regions flanking the deletion breakpoint is observed to increase. When the corresponding Hi-C reads are mapped to the reference genome, this increase in interaction frequency will be manifested in the spatial contact matrix heat map as a block of significantly darker color that can be observed in the interaction interval corresponding to the chromosome regions on both sides of the breakpoint. Similarly, any other form of chromosome structural change (including but not limited to duplication, insertion, translocation, inversion) will lead to an increase or decrease in the interaction frequency of the corresponding chromosome region, and the quantitative evaluation of this change is a key indicator for detecting whether there is structural variation in chromatin.

[0140] After establishing a sequencing library and obtaining sequencing data according to the method of the present invention, a genomic interaction matrix (e.g., a Hi-C contact map, for example, by juicer software) recording spatial contact information can be generated from the sequencing data according to methods known in the art to determine the type and position of structural variation and breakpoints on the sample to be tested.

[0141] In some cases, the processing of chromosome conformation capture sequencing data includes: sequence mapping, data filtering, and data correction. The reference genome used for sequence mapping can be any one of the human reference genomes GRCh37, GRCh38, T2T-CHM13, etc. For example, the PEreads sequence obtained by pair-ended sequencing can be compared with the reference genome to determine that only the paired reads that are uniquely matched to the reference sequence at both ends are obtained. After data filtering, the final valid data (i.e., valid contacts data) for contact analysis is obtained. The ratio of this valid contact data to all sequencing data is an important indicator for evaluating the quality of the chromosome conformation capture sequencing library. In some embodiments, the chromosome conformation capture library sequencing data obtained according to the method of the present invention has at least 50%, preferably at least 60% or 65% valid contacts.

[0142] In some embodiments, structural variation detection includes two main steps, namely (a) constructing a control reference system using healthy samples, and (b) performing variation detection on the sample to be tested. In some embodiments, structural variation detection is performed as shown in FIG2 .

[0143] In some embodiments, the variation detection of the sample to be tested includes the following steps:

[0144] (1) Convert the sequencing data into a numerical matrix that records spatial contact information, such as using Juicer

[0145] (https: / / doi.org / 10.1016 / j.cels.2016.07.002) The sequencing data were preprocessed and normalized to obtain the spatial contact value matrix, which contains the normalized contact positions and contact intensities;

[0146] (2) Correcting the spatial numerical matrix obtained in step (1), for example, in some embodiments, this step includes:

[0148] (i) Processing the spatial contact value matrix by quality control information. Preferably, in some embodiments, for each sample to be tested, the spatial contact matrix is ​​divided into bins of equal size of 1 Mb by rows and columns, and the interaction strength value between each pair of bins is recorded. The interaction strength of each bin is divided by the long range ratio of the sample to obtain the spatial contact strength value of the bin.

[0149] (ii) correcting the spatial contact value matrix using a matrix reference system constructed from the control reference system, preferably, in some embodiments, by subtracting the contact intensity baseline value at the corresponding position of the matrix reference system from each position of the value matrix; and

[0150] (iii) Convert the corrected spatial contact matrix to a different data format, such as by using HiSV ( https: / / doi.org / 10.1371 / journal.pcbi.1010760 )Convert the spatial contact information into a numerical matrix for subsequent data analysis;

[0151] (3) Inspecting chromosome structural variation on the standardized and corrected spatial contact matrix. In some embodiments, the inspection includes determining the position of the variation and identifying the direction of the variation. In other embodiments, the chromosome structural variation is a chromosome structural rearrangement and / or copy number variation, for example, selected from: translocation, balanced and unbalanced translocation, Robertsonian translocation, intra-arm and inter-arm inversion, insertion and CNV;

[0152] (4) Optionally, checking copy number variation (CNV) based on sequencing data and spatial contact value matrix;

[0153] (5) Optionally, the detected structural variation results are visualized and variation information is output.

[0154] In some embodiments, the test for balanced chromosomal translocation comprises the following steps:

[0155] (i) On a spatial contact matrix (e.g., a spatial contact matrix before correction using a matrix reference frame), e.g., by hic_breakfinder( https: / / github.com / dixonlab / hic_breakfinder and doi:10.1038 / s41588-018-0195-8), preliminarily calculating the positions of balanced translocation breakpoints;

[0156] (ii) On the corrected spatial contact matrix, based on the initially obtained balanced translocation breakpoint positions, the hypothetical breakpoint positions are set, and the difference index score is calculated based on the difference in contact density on both sides of the hypothetical breakpoint;

[0157] (iii) The position of the hypothetical breakpoint is stepped upstream and downstream respectively (for example, 5 Mb in steps of 100 kb), and the difference index score is recalculated for the new hypothetical breakpoint position obtained after each step, and the position of the highest difference index score that meets the following conditions at the end of the step is determined as the actual breakpoint position: if the highest score is higher than a preset score threshold (for example, the middle value of the highest difference index score obtained for samples that actually carry the translocation and the highest difference index score obtained for samples that do not carry the translocation), the breakpoint is judged to be positive and reported as the test result; otherwise, the breakpoint is judged to be a false positive and is not reported as the test result.

[0158] In some embodiments, the testing for balanced chromosomal translocation further comprises the following steps:

[0159] (iv) On the standardized and corrected spatial contact matrix, for each combination of two chromosomes, infer whether the centromere translocation hotspot region (for example, starting from the short arm side of the centromere and extending 5 Mb towards the short arm telomere, and the long arm side of the centromere extending 5 Mb towards the long arm telomere) meets the characteristics of balanced translocation (for example, the contact strength between the short arm of chromosome A and the short arm of chromosome B, and the contact strength between the long arm of chromosome A and the long arm of chromosome B, are both significantly greater than those between chromosome A and chromosome B).

[0160] For chromosome regions meeting this characteristic, the difference index score is inferred according to steps (i) to (iii) above, and the actual breakpoint position is calculated.

[0161] In some embodiments, the Robertson translocation test includes the following steps: on a standardized and corrected spatial contact matrix, for all combinations of chromosomes 13, 14, 15, 21, and 22, determine the spatial contact intensity in the matrix interaction interval corresponding to each chromosome combination and the difference in spatial contact intensity with other combinations, and calculate a difference index score; if the score is higher than a preset score threshold (for example, the middle value of the highest difference index score obtained for a sample that actually carries Robertson translocation and the highest difference index score obtained for a sample that does not carry Robertson translocation), it is determined that the corresponding chromosome combination has Robertson translocation, otherwise it is determined to be a false positive and is not reported as a test result.

[0162] In some embodiments, the chromosome structural abnormality inspection includes an inspection of inversion. In some embodiments, the inspection includes: searching for two matrix positions with high contact strength in the same chromosome on a standardized and corrected spatial contact matrix, and judging whether the position meets the inversion breakpoint characteristics (for example, the contact strength of breakpoint 1 upstream and breakpoint 2 upstream, breakpoint 1 downstream and breakpoint 2 downstream, are significantly greater than the contact strength of breakpoint 1 upstream and breakpoint 2 downstream, breakpoint 1 downstream and breakpoint 2 upstream, then judged as inversion); the position that meets the inversion breakpoint characteristics is inferred, and the difference index score is calculated. If the score is greater than the threshold, the breakpoint is determined as an inversion breakpoint.

[0163] In some embodiments, the chromosome structural abnormality inspection includes an inspection of insertion. The inspection includes: searching for matrix positions where the contact strength between different chromosomes is high on the standardized and corrected spatial contact matrix, judging whether it meets the insertion characteristics (for example, the contact strength between the fragment inside chromosome A and chromosome B is significantly enhanced), and also inferring the difference index score according to the above steps as a criterion for judging insertion variation.

[0164] In some embodiments, the present invention further provides a method for identifying the linked haplotype status of a structural variation in an embryo, the method comprising the steps of:

[0165] b1. Sampling: obtaining embryo biopsy cells and peripheral blood samples from both parents;

[0166] b2. using the method for identifying structural variation-linked haplotypes of the first aspect of the present invention, identifying structural variation-linked haplotypes of the structural variation carriers in the parents, and obtaining one or more breakpoint information related to the variation and two allele information of the linked SNP allele representing the structural variation haplotype and the linked SNP allele representing the normal haplotype;

[0167] b3. Extract DNA from embryo biopsy cells and peripheral blood samples of the non-carrier parent, amplify, build a library and then sequence;

[0168] b4. Align the genome sequences of the two samples measured in step b3 to the same human reference genome;

[0169] b5. For the sequence aligned to the reference genome in step b4, detect SNPs upstream and downstream of the breakpoints according to the mutation-related breakpoint information of the carrier;

[0170] b6. Intersecting the variant haplotype-linked SNP result obtained in step a10 of the method for identifying structural variation-linked haplotypes according to the first aspect of the present invention with the SNP result obtained in step b5 according to their positions on the genome, so as to infer which allele of the carrier is inherited by the embryo at that position, thereby independently determining for each SNP site of the embryo whether its genotype is consistent with the variant haplotype-linked SNP allele or consistent with the normal haplotype SNP allele;

[0171] b7. Count the number of SNP sites determined in step b6 upstream and downstream of the structural variation-related breakpoints. If the number of SNP sites consistent with the variant haplotype-linked SNP allele is greater than the number of sites consistent with the normal haplotype, then the embryo is considered to carry the structural variation haplotype obtained in step a10 of the first aspect, that is, it is presumed to carry the variation. Otherwise, it is presumed not to carry the variation.

[0172] In some embodiments, the biopsied cells of the embryo in step b1 are single or multiple cells from the blastocyst trophoblast. In some preferred embodiments, the blastocyst is a healthy and high-quality blastocyst cultured to day 5-8 after egg retrieval.

[0173] The following examples are described to assist understanding of the present invention. The examples are not intended to and should not be interpreted in any way as limiting the scope of protection of the present invention. Example

[0174] Example 1: Inference of balanced translocation

[0175] 1. Subject information: The female carries a balanced translocation 46,XX,t(7;20)(q31.32;p12.3), the male is 46,XY, and three embryos to be tested were obtained through in vitro fertilization.

[0176] 2. Obtain peripheral blood from the woman and the man, and extract genomic DNA (gDNA). The man used the Aowei Biotech TL674-03 pre-loaded magnetic bead method universal DNA extraction kit to cut and interrupt the library, and performed the MALBAC two-step method (Suzhou Xukang Medical Technology Co., Ltd.'s gene sequencing universal sample processing kit) whole genome amplification. The female carrier used Hi-C technology to build the library and then performed high-throughput sequencing.

[0177] 3. For the female Hi-C data, the sequences were aligned to the reference coordinate system hg19 using BWA (0.7.17-r1188), and the contact matrix was generated using juicer (2.0) (see Figure 1). Then, according to the matrix analysis, the breakpoints were obtained as chr7:121900000 and chr20:5300000, including 2 normal chain breakpoints (chr7:121900000; chr20:5300000) and 2 translocation variant chain breakpoints (chr7:121900000-chr20:5300000; chr20:5300000-chr7:121900000).

[0178] 4. For the female's Hi-C data, detect SNPs across the entire genome and retain heterozygous SNP results.

[0179] 5. The female's Hi-C data were screened according to the following conditions: the paired sequences spanned the normal chain breakpoint, one side was chr7:111900000-121900000 and the other side was chr7:121900000-131900000 or one side of the paired sequences was chr20:0-5300000 and the other side was chr20:5300000-15300000. SNPs were detected in the screened sequences within the above position range, and the homozygous SNP results were retained as the normal chain haplotype characteristic sites.

[0180] 6. The female's Hi-C data were screened according to the following conditions: the paired sequences spanned the translocation variant chain breakpoint, one side was chr7:111900000-121900000 and the other side was chr20:0-5300000 or one side of the paired sequences was chr7:121900000-131900000 and the other side was chr20:5300000-15300000. SNPs were detected for the screened sequences within the above position range, and the homozygous SNP results were retained as the variant chain haplotype characteristic sites.

[0181] 7. Take the intersection of the homozygous and heterozygous SNPs of the female carrier, retain the common sites, and determine the pathogenic chain. For example, at the position of chr7:121219538 (rs191194352), the pathogenic chain result is T / T, and the heterozygous result is T / G, which means that the SNPs corresponding to the two haplotypes at this site of the sample are T / G, the translocation haplotype corresponds to T, and the normal haplotype corresponds to C. Similarly, for the SNP corresponding to the normal chain, the pathogenic chain can also be determined based on the base on the opposite side of the heterozygous base pair.

[0182] Table 1 below shows some of the common sites by way of example (not all of them can be listed due to space limitations).

[0183] Table 1: Some effective SNP sites in Example 1

[0184]

[0185]

[0186] 8. For the female's Hi-C data, local haplotype blocks were constructed to obtain other SNPs linked to the identified normal / translocation SNPs (Table 1).

[0187] 9. Extract embryonic blastocyst trophoblast cells and perform single-cell whole genome amplification using the MALBAC two-step method (Suzhou Xukang Medical Technology Co., Ltd.'s universal sample processing kit for gene sequencing).

[0188] 10. Perform genotyping tests on the male's gDNA and embryo amplification products to obtain the genotype of specific SNP sites.

[0189] 11. Extract the SNP site information within 10M upstream and downstream of the mutation breakpoint, and select the sites where the mutation carrier is heterozygous and the normal spouse is homozygous, or the mutation carrier is heterozygous and the embryo is homozygous for the next step of analysis. The sites that meet the conditions are effective haplotype sites, and the carriers are distinguished into a series of variants and normal linked haplotypes according to step 6 (Table 1).

[0190] 12. After the haplotype construction is completed, the embryos can be divided into those carrying structural variant haplotypes and those carrying normal haplotypes according to the haplotypes carried by the embryos and the phenotypic information of their parents (see Figure 2a and Figure 2b Embryos 1 and 3 carry the maternal mutation (inherited dark brown haplotype), while embryo 2 does not carry the haplotype and carries the normal haplotype (inherited light brown haplotype).

[0191] 13. The embryo amplification products were also screened for aneuploidy using CNV-seq, and no abnormal chromosome copy number was found (Table 2).

[0192] 14. Further verification by family linkage analysis showed that embryos 1 and 3 carried balanced maternal translocations, while embryo 2 did not carry balanced maternal translocations, which was consistent with the results of family-free analysis (Table 2).

[0193] Table 2: Test results statistics in Example 1

[0194] Sample name Aneuploidy test results Structural variation detection results Embryo 1 46,XN Carrying translocation Embryo 2 46,XN Do not carry Embryo 3 46,XN Carrying translocation

[0195] Example 2: Inference of Robertsonian translocation

[0196] 1. Subject information: The female carries Robertsonian translocation 45,XX,der(13;14)(q10;q10), the male carries 46,XY, and there are four embryo samples to be tested.

[0197] 2. The same process as in Example 1 was followed for sampling, library construction, sequencing, and typing of the pedigree. When screening for homozygous SNPs on the carrier side, the paired sequences with one side at chr13:19400000-29400000 and the other side at chr14:20500000-30500000 were used as variant chains for SNP calling. There was no available normal chain SNP for Roche ectopic loci.

[0198] 3. According to the haplotype carried by the embryo and the phenotypic information of its parents, the embryo is divided into one carrying a structural variant haplotype and one carrying a normal haplotype ( Figure 3a and 3b Each shows a portion of the loci). Embryos 2, 3, and 4 carry the maternal mutation (inherited dark brown haplotype), while embryo 1 does not carry the haplotype and carries the normal haplotype (inherited light brown haplotype).

[0199] 4. The embryo amplification products were also screened for aneuploidy using CNV-seq, and no abnormal chromosome copy number was found (Table 3).

[0200] 5. Family linkage analysis verified that embryos 2, 3, and 4 carried maternal balanced translocations, while embryo 1 did not carry maternal balanced translocations, which was consistent with the results of non-family linkage analysis (Table 3).

[0201] Table 3: Test results statistics in Example 2

[0202] Sample name Aneuploidy test results Structural variation detection results Embryo 1 46,XN Do not carry Embryo 2 46,XN Carrying translocation Embryo 3 46,XN Carrying translocation Embryo 4 46,XN Carrying translocation

[0203] Example 3: Inference of inversion

[0204] 1. Subject information: The female carries the inversion 46,XX,inv(5)(p13.1q34), the male carries 46,XY, and there are five embryo samples to be tested.

[0205] 2. Sampling, library construction, sequencing, and typing were performed on the family according to the process of Example 1. For the female's Hi-C data, a contact matrix was generated using juicer ( Figure 4 ), and according to the matrix analysis of the variant breakpoints, the breakpoints chr5:40900000 and chr5:165200000 (the reference coordinate system is hg19) were obtained, including 2 normal chain breakpoints (chr5:40900000; chr5:165200000) and 2 inversion variant chain breakpoints (chr5:40900000-chr5:165200000; chr5:165200000-chr5:40900000).

[0206] 3. When screening homozygous SNPs on the carrier side, take the paired sequences with one side at chr5:35900000-40900000 and the other side at chr5:40900000-45900000, and the paired sequences with one side at chr5:160200000-165200000 and the other side at chr5:165200000-170200000 as the normal chain for SNP calling.

[0207] 4. When screening homozygous SNPs on the carrier side, take the paired sequences with one side at chr5:35900000-40900000 and the other side at chr5:160200000-165200000, and the paired sequences with one side at chr5:40900000-45900000 and the other side at chr5:165200000-170200000 as variant chains for SNP calling.

[0208] 5. According to the haplotype carried by the embryo and the phenotypic information of its parents, the embryo is divided into one carrying a structural variant haplotype and one carrying a normal haplotype ( Figure 5 ). Embryos 1, 2, 3, and 4 are carriers of the maternal mutation (inherited dark brown haplotype), while embryo 5 does not carry the haplotype and carries the normal haplotype (inherited light brown haplotype).

[0209] 6. The embryo amplification products were also screened for aneuploidy using CNV-seq, and no abnormal chromosome copy number was found (Table 4).

[0210] 7. Family linkage analysis verified that embryos 1, 2, 3, and 4 carried maternal inversions, while embryo 5 did not carry maternal inversions, which was consistent with the results of non-family linkage analysis (Table 4).

[0211] Table 4: Test results statistics in Example 3

[0212] Sample name Aneuploidy test results Structural variation detection results Embryo 1 46,XN Carrying inverted position Embryo 2 46,XN Carrying inverted position Embryo 3 46,XN Carrying inverted position Embryo 4 46,XN Carrying inverted position Embryo 5 46,XN Do not carry

[0213] Example 4: Inference of insertion variation

[0214] 1. Subject information: The female carries the insertion mutation 46,XX,ins(17;20)(q24.3;q11.22q13.13), the male is 46,XY, and there are three embryo samples to be tested.

[0215] 2. Sampling, library construction, sequencing, and typing were performed on the family according to the process of Example 1. For the female's Hi-C data, a contact matrix was generated using juicer ( Figure 6), and according to the matrix analysis of variant breakpoints, the insertion position breakpoint chr17:70400000, the insertion sequence breakpoints chr20:32800000 and chr20:47300000 (the reference coordinate system is hg19), including 1 normal chain breakpoint (chr17:70400000) and 2 insertion variant chain breakpoints (chr17:70400000-chr20:32800000; chr17:70400000-chr20:47300000).

[0216] 3. When screening for homozygous SNPs on the carrier side, take the paired sequence with one side located at chr17:60400000-70400000 and the other side located at chr17:70400000-80400000 as the normal chain for SNP calling.

[0217] 4. When screening homozygous SNPs for the carrier, take the paired sequences with one side at chr17:60400000-70400000 and the other side at chr20:32800000-42800000 or one side at chr20:37300000-47300000 and the other side at chr17:70400000-80400000 as the variant chain for SNP calling.

[0218] 5. According to the haplotype carried by the embryo and the phenotypic information of its parents, the embryo is divided into one carrying a structural variant haplotype and one carrying a normal haplotype ( Figure 7 ). Embryo 1 is a carrier of the maternal mutation (inherited dark brown haplotype), while embryos 2 and 3 do not carry this haplotype and carry the normal haplotype (inherited light brown haplotype).

[0219] 6. The embryo amplification products were also screened for aneuploidy using CNV-seq, and no abnormal chromosome copy number was found (Table 5).

[0220] 7. Family linkage analysis verified that embryo No. 1 carried maternal inversion, while embryos No. 2 and No. 3 did not carry maternal inversion, which was consistent with the results of non-family analysis (Table 5).

[0221] Table 5: Test results statistics in Example 3

[0222] Sample name Aneuploidy test results Structural variation detection results Embryo 1 46,XN Carry Insert Embryo 2 46,XN Do not carry Embryo 3 46,XN Do not carry

[0223] Example 5: Inference of deletion variants

[0224] 1. Since the copy number balanced insertion variation actually loses a sequence at the original position of the inserted fragment, it can be considered that the copy number balanced insertion variation also carries a deletion variation ( Figure 8 ). Under this premise, the deletion mutation carried by the female in Example 4 should be 46,XX,del(20)(q11.22q13.13).

[0225] 2. Sampling, library construction, sequencing, and typing were performed on the family according to the process of Example 4. For the female's Hi-C data, a contact matrix was generated using juicer ( Fig. 9 ), and according to the matrix analysis of variant breakpoints, the deletion variant breakpoints chr20:32800000 and chr20:47300000 (the reference coordinate system is hg19) were obtained, including 2 normal chain breakpoints (chr20:32800000; chr20:47300000) and 1 deletion variant chain breakpoint (chr20:32800000-chr20:47300000).

[0226] 3. When screening for homozygous SNPs on the carrier side, take the paired sequence with one side at chr20:22800000-32800000 and the other side at chr20:32800000-42800000 or one side at chr20:37300000-47300000 and the other side at chr20:47300000-57300000 as the normal chain for SNP calling.

[0227] 4. When screening for homozygous SNPs on the carrier side, take the paired sequence with one side located at chr20:22800000-32800000 and the other side located at chr20:47300000-57300000 as the variant chain for SNP calling.

[0228] 5. According to the haplotype carried by the embryo and the phenotypic information of its parents, the embryo is divided into one carrying a structural variant haplotype and one carrying a normal haplotype ( Fig.10 ). Embryo 1 is a carrier of the maternal mutation (inherited dark brown haplotype), while embryos 2 and 3 do not carry this haplotype and carry the normal haplotype (inherited light brown haplotype).

[0229] 6. The embryo amplification products were also screened for aneuploidy using CNV-seq, and no abnormal chromosome copy number was found (Table 6).

[0230] 7. Family linkage analysis verified that embryo No. 1 carried maternal inversion, while embryos No. 2 and No. 3 did not carry maternal inversion, which was consistent with the results of non-family analysis (Table 6).

[0231] Table 6: Test results statistics in Example 3

[0232]

[0233]

Claims

1. A method for identifying structural variation linkage haplotypes, the method comprising the steps of: a1) Obtain biological samples from structural variation carriers and extract genomic DNA; a2) constructing a library and sequencing the extracted genomic DNA using chromosome conformation capture technology; a3) aligning the genome sequence measured in step a2) to the human reference genome; a4) determining one or more structural variation breakpoints based on the sequence aligned to the reference genome in step a3) and combining the data characteristic signals obtained by sequencing using chromosome conformation capture technology; a5) for the sequence aligned to the reference genome in step a3), detecting SNPs upstream and downstream of the one or more breakpoints according to the one or more breakpoint information obtained in step a4), and retaining heterozygous SNP results; a6) extracting the paired-end sequencing information of the sequences aligned to the reference genome in step a3), and screening them according to their genomic positions, wherein the screening criteria are to retain the paired-end sequences whose two ends can be respectively aligned to the upstream and downstream of the breakpoint positions corresponding to the normal chromosomes without mutation as the normal chain characteristic sequences, and to retain the paired-end sequences whose two ends can be respectively aligned to the upstream and downstream of the breakpoints of the newly generated chromosomes after mutation as the variant chain characteristic sequences, and to remove the sequences that do not meet the above criteria; a7) detecting SNPs in the sequences retained in step a6), and retaining homozygous SNP results; a8) taking the intersection of the SNP sites retained in step a5) and the SNP sites retained in step a7), and comparing the effective depths of each site in the intersection in step a5) and step a7), retaining the site with an effective depth greater than that in step a7) in step a5) as a haplotype anchor point, wherein at this site, the homozygous base obtained in step a7) is a characteristic base representing the structural variation haplotype, and the other of the heterozygous bases obtained in step a5) is a characteristic base representing a normal chromosome; a9) assembling and constructing local linked SNP blocks for the sequences aligned to the reference genome in step a3); a10) In the local linked SNP block obtained in step a9), search for the haplotype anchor position obtained in step a8). For the block containing the anchor SNP, use the base detected in step a8) as a reference, and use the SNP allele linked to the base as the linked SNP allele representing the structural variation haplotype, and the other as the linked SNP allele representing the normal haplotype.

2. The method of claim 1, wherein the chromosome conformation capture technology in step a2) is Hi-C technology.

3. The method of claim 1, wherein the reference genome in step a3) is hg19, hg38, T2T, etc.

4. The method of claim 1, wherein the one or more structural variation breakpoints detected in step a4) include normal chain breakpoints and / or variant chain breakpoints.

5. The method of claim 4, wherein the variant chain breakpoints include variant position breakpoints and / or variant sequence breakpoints.

6. The method of claim 1, wherein: In step a3), the genome alignment is performed using genome alignment tool software, preferably, the software is BWA, samtools, Bowtie2, HiC-Pro and / or hisat2; In step a4), the breakpoints are detected by Hi-C data analysis software, preferably, the software is juicer; The software used for detecting SNP in step a5) is GATK or freebayes; and / or In step a9), haploid assembly analysis software is used to assemble and construct local linkage SNP blocks. Preferably, the software is HapCUT or HapCUT2, more preferably HapCUT2.

7. The method of claim 1, wherein: The upstream and downstream range of the breakpoint in step a5) is preferably 10 Mbp upstream and downstream of a balanced translocation breakpoint, 10 Mbp downstream of a Roche ectopic breakpoint, 5 Mbp upstream and downstream of an inversion breakpoint, and 10 Mbp upstream and downstream of an insertion mutation breakpoint; and / or Step a6) The range upstream and downstream of the breakpoint is 10 Mbp upstream and downstream of the balanced translocation breakpoint, 10 Mbp downstream of the Roche ectopic breakpoint, 5 Mbp upstream and downstream of the inversion breakpoint, and 10 Mbp upstream and downstream of the insertion mutation breakpoint.

8. The method of claim 1, wherein the structural variation is selected from the group consisting of structural rearrangements and copy number abnormalities (CNVs), preferably structural rearrangements, more preferably the structural rearrangement structural variation is selected from the group consisting of translocations (balanced translocations, unbalanced translocations, reciprocal translocations, non-reciprocal translocations, Robertsonian translocations, and complex translocations), inversions (intra-arm inversions and inter-arm inversions), and insertions (forward insertions and inversion insertions).

9. The method of claim 1, wherein the structural variation comprises a copy number abnormality (CNV), and the copy number abnormality structural variation is selected from a deletion and a duplication.

10. The method of claim 1, wherein the structural variation is selected from chromosome structural changes with sequence breakpoints such as isochromosomes and ring chromosomes.

11. The method of claim 1, wherein the sequencing is low-depth double-end sequencing, preferably, the sequencing depth is not less than 2X and / or not more than 10X, more preferably, the sequencing depth is not less than 3X and / or not more than 5X, about 30M reads to about 40M reads, preferably, the double-end sequencing is double-end paired sequencing.

12. The method of claim 1, wherein the structural variation carrier has no genetic information of other family members.

13. A method for identifying the linkage haplotype status of a structural variation in an embryo, the method comprising the steps of: b1) Sampling: obtaining embryo biopsy cells and peripheral blood samples from both parents; b2) using the method of any one of claims 1 to 12 to identify the structural variation-linked haplotype of the structural variation carrier in the parents, and obtaining one or more breakpoint information related to the variation and two allele information of the linked SNP allele representing the structural variation haplotype and the linked SNP allele representing the normal haplotype; b3) DNA was extracted from peripheral blood samples and embryo biopsy cells of the non-carrier parent of both parents, and amplified, library-built and then sequenced; b4) aligning the genome sequences of the two samples measured in step b3) to the same human reference genome; b5) for the sequence aligned to the reference genome in step b4), detecting SNPs upstream and downstream of the breakpoints according to the mutation-related breakpoint information of the carrier; b6) taking the intersection of the variant haplotype-linked SNP result obtained in step a10) of the method of claims 1-12 and the SNP result obtained in step b5) according to their positions on the genome to infer which allele of the carrier is inherited by the embryo at that position, thereby independently determining for each SNP site of the embryo whether its genotype is consistent with the variant haplotype-linked SNP allele or consistent with the normal haplotype SNP allele; b7) counting the number of SNP sites determined in step b6) upstream and downstream of the structural variation-related breakpoints, and if the number of SNP sites consistent with the variant haplotype-linked SNP allele is greater than the number of sites consistent with the normal haplotype, then The embryo is considered to carry the structural variation haplotype obtained in step a10) of the method of claims 1-12, that is, it is presumed to carry the variation; otherwise, it is presumed not to carry the variation.

14. The method of claim 13, wherein the biopsied cells of the embryo in step b1) are single or several cells from the trophoblast of a blastocyst, and preferably the blastocyst is a blastocyst cultured to day 5-8.

15. A detection product for implementing the method according to any one of claims 1 to 14, comprising one or more of the following modules: (1) gDNA extraction module: used to extract genomic DNA from a sample (e.g., a cell); (2) Chromosome conformation capture module: used to fix the chromosome conformation in the sample, that is, to fix the interactions between spatially contacting or adjacent DNA fragments and / or with proteins, for example, by covalent cross-linking, so that they can be detected in subsequent steps; (3) Sequencing library construction module: used to generate a whole genome DNA sequencing library; (4) Sequencing module: used for low-depth double-end sequencing of the sequencing library; (5) Alignment module: used to align the whole genome sequencing results generated by the sequencing module to the reference genome; (6) Structural variation assessment module: used to assess the chromosome structural variation of sample genomic DNA from sequencing data; (7) SNP detection module: used to detect SNP sites and their genotypes from sequencing data; (8) Typing module: used to compare and screen related SNP sites and identify haplotype-related linked SNP alleles.

16. The detection product of claim 15, which is a detection system.

17. The product of claim 15 or 16 for use in carrying out the method of any one of claims 1 to 14.

18. Use of the method according to any one of claims 1 to 14, or the product according to claim 15 or 16, for pre-embryo implantation screening and / or prenatal screening, or for the preparation of a product for pre-embryo implantation screening and / or prenatal screening.

Images