A method for rapid parentage identification by polymorphic locus linkage block

Abstract

The application relates to a method for rapid parentage identification by linkage block of polymorphic loci and application thereof, and the like. The method comprises the following steps: establishing a linkage database of polymorphic loci, obtaining low-depth WGS sequencing data of a sample, obtaining a mapping relationship between a point locus and linkage polymorphism, and performing parentage identification according to the low-depth WGS sequencing data and the mapping relationship between the point locus and the linkage polymorphism and taking a plurality of loci constituting the linkage polymorphism as a unit. The method has a short cycle, can realize parentage identification results within 24 hours without specific regional polymorphic locus capture, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological information analysis, and particularly relates to a method for rapid parentage identification by linkage block of polymorphic sites. BACKGROUND

[0002] Parentage identification refers to the identification of the blood relationship between individuals through the detection of human genetic markers and the analysis according to genetic rules. Non-invasive prenatal parentage identification refers to the extraction of peripheral blood from a pregnant woman, the sequencing of free DNA fragments therein, the acquisition of fetal DNA information, the comparison with the DNA information of the parents, the parentage identification through Mendelian inheritance law, and the like.

[0003] Parentage identification types include trios parentage identification (Parentage Testing of Trios), which refers to the parentage identification of a detected man, the child's mother and the child, or the parentage identification of a detected woman, the child's father and the child; and duos parentage identification (Parentage Testing of Duos), which refers to the parentage identification of a detected man and the child, or the parentage identification of a detected woman and the child.

[0004] According to the requirements of the Technical Specification for Parentage Identification (GB / T 37223-2018), after obtaining the genotype results of the parents / parent and the child, it is necessary to calculate the parentage index (PI value) and summarize the PI values of various sites to obtain the cumulative parentage index (CPI value). Only when the CPI value is greater than 10000 can it support the existence of a kinship relationship. If the parents / parent and the child do not have a kinship relationship, a phenomenon that multiple loci do not comply with Mendelian inheritance law will occur.

[0005] For example, patent No. CN202010366838.8 discloses a non-invasive prenatal parentage detection and analysis method, which comprises the following steps: obtaining target region sequencing data of free DNA in peripheral blood of a to-be-tested pregnant woman and genomic DNA target region sequencing data of both parents of a fetus, wherein the free DNA in peripheral blood of the to-be-tested pregnant woman comprises free DNA of the to-be-tested pregnant woman herself and free DNA of the fetus; analyzing the sequencing data to determine the genotypes of a plurality of genetic marker sites of the to-be-tested pregnant woman and both parents of the fetus; inferring the genotypes of the fetus at the plurality of genetic marker sites by using the difference between the variation detection result of the peripheral blood of the pregnant woman and the base distribution of the free DNA; calculating the cumulative parentage index CPI and / or the cumulative exclusion probability CPE by using trios or duos parentage identification method according to the genotypes of the to-be-tested pregnant woman, both parents of the fetus and the fetus at the plurality of genetic marker sites; and judging the kinship relationship between the to-be-tested pregnant woman or both parents of the fetus and the fetus according to the cumulative parentage index CPI and / or the cumulative exclusion probability CPE.

[0006] For the sequencing method used in parentage testing, the existing ngs data used for prenatal non-invasive parentage testing is basically high-depth sequencing, using hybridization probes to capture specific region information, and the sequencing depth is usually above 200X. WGS sequencing is not specific to a certain region, and covers the whole genome. If high-depth sequencing is obtained, the sequencing cost is very high, for example, 30X wgs human genome sequencing requires about 90G of base sequencing, and the current sequencing cost is about 2000 yuan. Therefore, wgs sequencing has an advantage over hybridization, reducing the operation process and time-consuming. Low-depth wgs data typing has low reliability, and cannot directly indicate the fetal signal in the maternal to separate. Therefore, how to use low-depth wgs data for parentage testing is a technical problem that needs to be solved in the field.

[0007] In the prior art, additional steps are needed to enrich polymorphic sites, increasing the complexity of the operation and additional time, resulting in a longer time to obtain the results of parentage testing. In order to be more widely used in industry, the field urgently needs to provide a method for parentage testing that can quickly enrich polymorphic sites without additional experimental steps. SUMMARY

[0008] In view of the deficiencies of the prior art, the purpose of the present application is to provide a method for rapid parentage testing by polymorphic site linkage block, which is based on low-depth WGS sequencing method for parentage testing and can quickly identify.

[0009] The present application is based on the following principle: SNP (Single Nucleotide Polymorphism) is a common form of genetic variation, which exists widely in human genome. Linkage disequilibrium (LD) is used to describe the non-random association between two or more genetic markers, usually SNPs. When the combination of alleles of certain SNPs appears more frequently in a population than expected from the individual allele frequencies, this phenomenon is called linkage disequilibrium. D is the basic unit of LD (linkage disequilibrium), which measures the deviation of the observed block frequency from the expected frequency under the equilibrium state. LD can be measured from 0-1, the higher the LD, the stronger the linkage between two sites if they are linked. The present application uses linkage SNP blocks as the calculation unit, which can equivalently increase the depth.

[0010] One aspect of the present application provides a method for rapid parentage testing by polymorphic site linkage block, characterized in that the method comprises the following steps:

[0011] S1: establishing a linkage relationship database of polymorphic sites;

[0012] S2: performing low-depth WGS sequencing on the sample according to the requirements of parentage testing to obtain sequencing data;

[0013] S3: filtering the typing data of the corresponding sites constituting a linkage polymorphic site region block on the reference genome in the sequencing data according to the linkage relationship of the polymorphic sites;

[0014] S4: performing parentage identification in units of linkage polymorphic site region blocks according to the typing data;

[0015] The linkage polymorphic site region block is a linkage polymorphic site region block composed of a plurality of linkage sites with high linkage degree, and the reference genome is public genome WGS data of a corresponding population or population.

[0016] Further, the polymorphic sites include SNPs, microsatellite markers, insertion / deletion polymorphisms, and copy number variations; preferably, the polymorphic sites are SNPs.

[0017] Further, in step S1, the linkage relationship of the polymorphic sites is obtained in at least one of the following ways: (1) obtained by processing public genome WGS data; (2) obtained by processing a large amount of high-depth WGS measured data; (3) obtained from haploid cells such as germ cell sequencing data; (4) obtained by processing long sequencing mode such as sanger, third-generation sequencing data; 5) obtained by querying public databases and literature.

[0018] Further, wherein the linkage relationship of the polymorphic sites is obtained by processing public genome WGS data or the linkage relationship of the polymorphic sites is obtained by processing a large amount of high-depth WGS measured data, specifically obtained by screening based on a reference genome according to a predetermined rule.

[0019] Preferably, the predetermined rule is selected from any one of the following:

[0020] (1) taking SNPs from the reference genome and removing sites and regions that do not meet the requirements, calculating the LD value between the SNP sites, and selecting the region block with a specific range of population frequency as the linkage SNP region block;

[0021] (2) isolating gametes of a specific population to obtain region blocks, and further obtaining heterozygous region block data;

[0022] (3) selecting a plurality of individuals in the population for sequencing to obtain region block sequencing data with a fragment length of >50kb in the whole genome range, and further obtaining heterozygous region block data;

[0023] (4) using the public database LDLink, screening the region blocks of a specific population through the LDhap of the LDLinkR software package, and further obtaining heterozygous region block data.

[0024] Further, the predetermined rule is (1), which specifically comprises the following steps:

[0025] (1) Take SNPs with population frequency between 0.2-0.8 from autosomes of the reference genome from G WGS data, and remove sites and regions that do not meet the requirements, preferably G is greater than 200;

[0026] (2) On a chromosome, calculate sequentially from front to back, fix the first SNP site as SNP1, calculate the LD value between the subsequent SNP site and SNP1, and count these values until at least K LD values in the continuous M LD values are < the first threshold T1 cutoff, and SNP1 to the cutoff site is the first regional block;

[0027] (3) Screen the SNP sites in the first regional block, and take the SNP sites with LD values greater than the second threshold T2 as linkage sites, and the linkage sites in the first regional block form the second regional block; Then judge the second regional block corresponding to the regional block, if P(hap1)+P(hap2)>T3, then the second regional block is retained; otherwise, the second regional block is deleted; wherein hap1 and hap2 are the two most frequent heterozygous regional blocks in the second region, P(hap1) is the frequency of regional block hap1 appearing in G data, and T3 is the third threshold;

[0028] (4) In this way, the subsequent adjacent linkage sites are fixed, and then the LD values are calculated with the subsequent SNP sites, and all second regional blocks on the chromosome are obtained according to steps (2)-(3); sort all second regional blocks according to the number of linkage sites, and only keep the second regional blocks with LD values between the second regional blocks less than the fourth threshold T4;

[0029] (5) In this way, the second regional blocks of multiple chromosomes are obtained according to steps (2)-(4);

[0030] (6) Screen the second regional blocks of the multiple chromosomes, and select the second regional blocks with population frequency between 0.2-0.8 as linkage SNP regional blocks.

[0031] Further, the predetermined rule is (2), specifically comprising the following steps: obtaining a region block of gametes of a specific population: selecting semen of 400-1000 individuals in the population, obtaining single sperm, performing single-cell second-generation sequencing to obtain sequencing data; aligning the sequencing data to a reference genome to obtain data of 400-1000 region blocks; obtaining frequency data of independent polymorphisms of the population by aligning the data of 400-1000 region blocks; taking SNPs with a frequency of 0.2-0.8 in the population and removing sites and regions that do not meet the requirements; taking each SNP as a starting point, expanding all SNPs within 100 kbp to form a linkage group of the SNP, and screening the SNP linkage group by the following conditions: the sum of the frequencies of a specific region block P and its alien region block Q in the linkage group is greater than 0.95, and the region block covers more than N SNPs, wherein N is an integer greater than or equal to 2, preferably N is an integer of 10-30; the specific region block P includes a missing region block; if a region block is AAAaaa, then aaaAAA is its alien region block, and A-Aaaa and AAA--a are its missing region blocks.

[0032] Further, the predetermined rule is (3), selecting 400-1000 individuals in the population, obtaining sequencing data of region blocks with a whole-genome range fragment length of >50 kb by third-generation sequencing; aligning the sequencing data to a reference genome to obtain data of 400-1000 region blocks; obtaining frequency data of independent polymorphisms of the population by aligning the data of 400-1000 region blocks; taking SNPs with a frequency of 0.2-0.8 in the population and removing sites and regions that do not meet the requirements; taking each SNP as a starting point, expanding all SNPs within 100 kbp to form a linkage group of the SNP, and screening the SNP linkage group by the following conditions: the sum of the frequencies of a specific region block P and its alien region block Q in the linkage group is greater than 0.95, and the region block covers more than N SNPs, wherein N is an integer greater than or equal to 2, preferably N is an integer of 10-30; the specific region block P includes a missing region block; if a region block is AAAaaa, then aaaAAA is its alien region block, and A-Aaaa and AAA--a are its missing region blocks.

[0033] Further, the predetermined rule is (4), using the public database LDLink, screening the specific population's regional block by LDhap of the LDLinkR software package: the sum of the frequencies of a certain specific regional block P and its full block Q within the linkage group is greater than 0.95, and the regional block covers more than N SNPs, wherein N is an integer greater than or equal to 2, preferably N is an integer of 10-30; the specific regional block P includes a missing block; if a certain block is AAAaaa, then aaaAAA is its full block, A-Aaaa and AAA--a are its missing blocks.

[0034] Further, in the unit of linkage SNP regional block: align the linkage SNP regional block as a whole with the reference genome, and genotype according to the sequencing depth ratio, the sequencing depth of the linkage SNP regional block is the superposition of the sequencing depth of the corresponding genotyped linkage site within it.

[0035] Further, in the step S1, the sample is a pregnant woman's free sample S, a maternal leukocyte sample M, and a sample F to be tested; in the step S3, according to the genotyping of the linkage SNP regional block and the population frequency, the genotype of the fetus is obtained by the sequencing depth ratio of the linkage SNP regional block of S and M, and the genotype of the father is obtained by the sequencing depth ratio of the linkage SNP regional block of F; the parentage identification is performed by calculating the error rate and CPI.

[0036] Further, the process of obtaining the genotype of the fetus is: if the sequencing depth ratio of S at the site is 0.01

[0037] Further, the parentage identification includes but is not limited to parent-child relationship, sibling relationship, half-sibling relationship, uncle-niece relationship, grandparent-grandchild relationship, or great-grandparent-great-grandchild relationship.

[0038] Further, the parentage identification includes non-invasive prenatal parentage identification.

[0039] Further, the method is suitable for diploid organisms complying with Mendelian inheritance; preferably, the method is suitable for humans; more preferably, the method is suitable for Chinese population; but not as a limitation, the method is also suitable for other animals (such as monkeys), other human populations (such as African population).

[0040] The method for parentage identification by linkage block of polymorphic sites according to the present application has the following beneficial technical effects:

[0041] 1. The method according to the present application does not require obtaining genotyping data of independent polymorphic sites, and does not require a step of enriching polymorphic sites, thereby achieving convenient and rapid obtaining of parentage identification results.

[0042] 2. The method according to the present application has a short cycle, and can realize obtaining parentage identification results within 24 hours without capturing specific region polymorphic sites, thereby having a wide application prospect.

[0043] 3. The linkage SNP region block utilized by the present application has a high linkage disequilibrium (LD), and has a strong genetic stability and is less affected by random variation. Calculating in units of linkage SNP region block can effectively reduce random error and improve the stability and reliability of the results.

[0044] 4. The present application can accurately determine parentage relationship in low-depth WGS data by optimizing the algorithm and analyzing the linkage SNP region block, thereby reducing the cost and maintaining a high accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 The flow chart of the method for rapid parentage identification by linkage block of polymorphic sites according to the present application is provided.

[0046] Figure 2 The control relationship diagram of base position and LD value of chr4:213204 site in the method of the embodiment.

[0047] Figure 3 The mapping relationship diagram between the number of fetal information points and CPI value in the method of the embodiment. DETAILED DESCRIPTION

[0048] TERMS AND DEFINITIONS

[0049] The term "genomic WGS data (Whole Genome Sequencing Data)" refers to genomic data obtained by whole genome sequencing technology, which contains sequence information of all bases in the genome.

[0050] The term“low depth WGS (Whole Genome Sequencing)” refers to whole genome sequencing of a sample, but at a lower depth of sequencing, which is not sufficient to genotype the sample by the data of polymorphic sites.

[0051] The term“Cell-Free Fetal DNA (cffDNA)”: refers specifically to the fetal-derived cell-free DNA fragments in the blood of a pregnant woman, which can be used for non-invasive prenatal testing (NIPT).

[0052] The term“Linkage Polymorphism” refers to a combination of multiple sites in the genome that tend to be transmitted together during inheritance. This combination of polymorphisms presented by multiple sites is called linkage polymorphism. In the present patent method, the calculation of fetal DNA concentration is carried out in units of multiple sites that form linkage polymorphism relationships.

[0053] The term“sequencing depth” refers to the average number of times each base is sequenced during sequencing. Low-depth sequencing generally refers to a smaller number of times each base is sequenced.

[0054] The term“reference genome GRCh38 (Genome Reference Consortium Human Build 38)” is a standard human genome reference sequence version released by the Human Genome Project. It is one of the widely used reference genomes at present, maintained and updated by the Genome Reference Consortium (GRC).

[0055] The term“SNP region block” refers to a unit set consisting of multiple linkage SNPs, including a core SNP site and one or more SNP sites linked to it.

[0056] The term“linkage region block” refers to a unit set consisting of multiple polymorphic genetic marker sites with linkage relationships, including a core polymorphic site and one or more polymorphic sites linked to it.

[0057] Unless otherwise defined or indicated by the context, all technical and scientific terms in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

[0058] For low-depth wgs data typing credibility is low, and the technical problem of directly indicating the separation of fetal signals in the maternal inside cannot be solved, the present application provides a solution to mine high-depth genetic variation types in wgs sequencing data, judge the genotype of the mother, separate the fetal signal, and be used for concentration evaluation, parentage identification, etc.

[0059] The present application explores the relationship between genetic variations, which are exemplified by SNPs below.

[0060] For example, two sites in the CHB, CHS population of the 1000 Genomes Project,

[0061] chr4:213231:G>A rs5003260

[0062] chr4:221985:G>A rs6830496

[0063] The assembled genome has the following rules:

[0064] a. When the base at chr4:213231 is detected as G, G must be detected at chr4:221985.

[0065] b. When the base at chr4:213231 is detected as A, A must be detected at chr4:221985.

[0066] Or under the condition of not being good to assemble, the typing results also have the following rules:

[0067] a. When the genotype at chr4:213231 is detected as GG, GG must be detected at chr4:221985.

[0068] b. When the genotype at chr4:213231 is detected as AA, AA must be detected at chr4:221985.

[0069] c. When the genotype at chr4:213231 is detected as AG, AG must be detected at chr4:221985.

[0070] This situation is extended to the same rule in third-generation sequencing, assembly sequencing, and reproductive cell single-cell sequencing results.

[0071] Further, it is found that the same is true at positions such as chr4:226360:T:C:rs11248009 and chr4:228314:C:T:rs61794997.

[0072] Furthermore, when performing WGS sequencing on a sample of a certain type of free blood, and finding the following base counting results, it can generally be concluded that at the position chr4:213231, although only 1X sequencing is achieved, based on the SNP information of the continuous linkage, the equivalent depth of chr4:213231 can be obtained as 4X, and the ro:ao ratio is 2:2.

[0073] chrom pos rsid ref alt dp ro ao chr4 213231 rs5003260 G A 1 1 0 chr4 221985 rs6830496 G A 1 0 1 chr4 226360 rs11248009 T C 1 1 0 chr4 228314 rs61794997 C T 1 0 1

[0074] Based on the potential patterns of the above genetic loci, the human genome is explored to find ways to effectively increase WGS sequencing depth. Various methods and algorithms exist to obtain and apply these patterns, such as using publicly available genomic information from the 1000 Genomes Project to calculate correlations and linkage equilibrium strength. The following is an example of LD calculation distribution based on chr4:213204:rs4624608. Figure 2 As shown, each bar represents the linkage strength between the SNP and chr4:213204:rs4624608. The horizontal axis can be seen as the chromosome, representing the distance of the SNP from chr4:213204:rs4624608 by the number of bases. Different colors correspond to different LD values. Due to the large distances between the bars, bars that are relatively close may merge together.

[0075]

[0076] Equivalent depth limitation

[0077] a. If the DNA content extracted from the sample is limited, such as only two genomes from one cell, the equivalent depth may exceed 2X, but it cannot be determined as a repetitive sequence. The result can be used for typing, but not for screening small signals.

[0078] b. Due to the selection of the target population and the biological errors inherent in the data source itself, maternal white blood cell data can be used to assist in the judgment when conducting prenatal non-invasive paternity testing.

[0079] Furthermore, this invention specifically provides a method for rapid paternity testing using polymorphic locus linkage regions. Figure 1 A flowchart of a method for rapid paternity testing using polymorphic site linkage blocks provided by an embodiment of the present invention is given as an example.

[0080] Specifically, the method includes the following steps:

[0081] S1: Establish a linkage database for polymorphic sites;

[0082] S2: Perform low-depth WGS sequencing on the sample according to the requirements of paternity testing to obtain sequencing data R;

[0083] S3: screening the typing data Q of the corresponding sites constituting the linkage SNP region block on the reference genome in the sequencing data R according to the linkage relationship of the polymorphic sites;

[0084] S4: performing parentage identification according to the typing data B and in units of linkage polymorphic site region blocks;

[0085] The linkage polymorphic site region block is a linkage polymorphic site region block composed of a plurality of linkage sites with high linkage degree, and the reference genome is public genome WGS data of a corresponding population or population.

[0086] Specifically, taking a linkage SNP region block as an example, but not as a limitation of the present application; the method for rapid parentage identification by linkage region block of polymorphic sites disclosed in the present application comprises the following steps:

[0087] S1: performing low-depth WGS sequencing on the sample according to the requirements of parentage identification, to obtain sequencing data R. The parentage identification of the present application includes but is not limited to parent-child relationship, sibling relationship, half-sibling relationship, uncle-niece relationship, grandparent-grandchild relationship or great-grandparent-great-grandchild relationship, etc. The present application takes non-invasive prenatal parentage identification as an example for illustration, but not as a limitation of the present application.

[0088] S2: screening the typing data Q of the corresponding sites constituting the linkage SNP region block on the reference genome in R according to the mapping relationship between the sites and the linkage SNP region block.

[0089] S3: performing parentage identification according to the typing data Q and in units of linkage SNP region blocks. The method for parentage identification can utilize the existing methods for parentage identification by typing, including but not limited to conventional triad parentage identification or diad parentage identification, etc. Different methods will result in different samples in step S1. The present application uses a pregnant woman's free sample S, a maternal white blood cell sample M and a sample F to be tested for the father, but not as a limitation of the present application.

[0090] The mapping relationship between the point and the linkage SNP region block is obtained by screening the reference genome according to a predetermined rule. The linkage SNP region block of the present application is a linkage SNP region block composed of a plurality of linkage sites with high linkage degree. According to a specific algorithm, the linkage sites in the linkage SNP region block have a long distance and meet specific conditions. When parentage identification is performed, the sequencing depth of the linkage SNP region block is the superposition of the sequencing depths of the corresponding typing linkage sites in the linkage SNP region block, which simplifies the processing process of the sequencing data and further accelerates the process of parentage identification. The reference genome is the public genome WGS data corresponding to the population or population. Of course, the reference genome can be obtained from the present application. The method of the present application can be applied to diploid organisms that meet Mendelian inheritance. The present application is illustrated by humans (Chinese population), but is not limited thereto. The method of the present application can be used for other animals (such as monkeys) and other populations (such as African populations).

[0091] The predetermined rule is:

[0092] (1) Take the SNP with a population frequency of 0.2-0.8 from the G WGS data of the autosome of the reference genome and remove sites and regions that do not meet the requirements.

[0093] (2) On a chromosome, calculate sequentially from front to back, fix the first SNP site as SNP1, calculate the LD value between the subsequent SNP site and SNP1, and count these values until at least K LD values in the continuous M LD values are less than the first threshold T1, and the SNP1 to the cutoff site is the first region block. This step can reduce the amount of calculation. Optionally, step (2) is not performed, and the subsequent steps can also be performed directly.

[0094] (3) The SNP sites in the first region block are screened, and the SNP sites with an LD value greater than the second threshold T2 are taken as linkage sites. The linkage sites in the first region block form a second region block. The second region block is judged again. If P(hap1)+P(hap2)>T3, the second region block is retained; otherwise, the second region block is deleted; wherein hap1 and hap2 are the two most different region blocks with the highest frequency in the second region, P(hap1) is the frequency of the region block hap1 in the G data, and T3 is the third threshold.

[0095] (4) In this way, the subsequent adjacent linkage sites are fixed, and the LD values are calculated with the subsequent SNP sites in turn. According to steps (2) and (3), all second region blocks on the chromosome are obtained. The second region blocks are sorted according to the number of linkage sites, and only the second region blocks with an LD value less than T4 between the second region blocks are retained to remove the intersection part, and T4 represents the fourth threshold.

[0096] (5) Similarly, the second region blocks of multiple chromosomes are obtained according to steps (2)-(4).

[0097] (6) The second region blocks are screened, and the second region blocks with a population frequency in a specific range are selected as the linkage SNP region blocks.

[0098] In the removal of the sites and regions that do not meet the requirements, the indel type sites are removed, the STR-related SNP sites are removed, the MSI-related SNP sites are removed, the Hardy-Weinberg equilibrium points are removed, the regions with the possibility of duplication are removed, and the regions with high GC content and low complexity near the SNP are removed, so as to reduce the amount of calculation and reduce the interference on the subsequent program.

[0099] In the formula, M is greater than or equal to 10, M-K is 1-4, T1 is 0.01-0.20, T2 is 0.90-0.99, T3 is 0.95-0.99, and T4 is 0.05-0.2. The specific range in step (6) can be 0.3-0.7. M, K, T1, T2, T3, T4 and the specific range can be adjusted according to the purpose, precision or platform, etc. Preferably, M is 12, K is 10, T1 is 0.1, T2 is 0.95, T3 is 0.98, and T4 is 0.1.

[0100] Specifically, in step (1), one or more populations of CHS, CHB and CDX in 1KGP are used as the reference genome. More specifically, 718 WGS data of autosomes of three populations of CHS, CHB and CDX in 1KGP are used, and of course 416 WGS data of autosomes of two populations of CHS and CHB in 1KGP can also be used. If it is used for populations in other countries, population data of the corresponding country is used. The number of SNPs of 22 pairs of chromosomes obtained by CHS and CHB is shown in Table 1.

[0101] Table 1 Number of SNPs of 22 pairs of chromosomes obtained by CHS and CHB

[0102] chromosome chrom SNP num chr1 243117 chr2 248355 chr3 215927 chr4 231182 chr5 197186 chr6 211535 chr7 186055 chr8 173553 chr9 135534 chr10 157705 chr11 156941 chr12 147287 chr13 115835 chr14 102990 chr15 91245 chr16 97307 chr17 80846 chr18 91798 chr19 72402 chr20 70515 chr21 46598 chr22 43084

[0103] Specifically, the predetermined rules are:

[0104] (1) 718 WGS data of autosomes of three populations of CHS, CHB and CDX in 1KGP are used, and SNPs with a population frequency of 0.2-0.8 are taken and indel type sites are removed, STR-related SNP sites are removed, MSI-related SNP sites are removed, Hardy-Weinberg equilibrium points are removed, regions with the possibility of duplication are removed, and regions with high GC content and low complexity near the SNP are removed.

[0105] (2) On a chromosome, calculate the LD value between the first SNP SNP1 and the subsequent SNP sites in sequence from front to back, and count these values until at least 10 of the 12 consecutive LD values are <0.1 cutoff, and the SNP1 to the cutoff site is the first region block.

[0106] (3) Screen the SNP sites in the first region block, and the SNP sites with LD values greater than 0.95 are the linkage sites, and the linkage sites in the first region block form the second region block; then judge the region block corresponding to the second region block, if hap1+hap2>718*0.98, the second region block is retained; otherwise, the second region block is deleted.

[0107] (4) In this way, the subsequent adjacent linkage sites are fixed, and the LD values are calculated with the subsequent SNP sites in sequence, and the second region blocks on the chromosome are obtained according to steps (2) and (3); the second region blocks are sorted according to the number of linkage sites, and only the second region blocks with LD values less than 0.1 between the second region blocks are retained.

[0108] (5) In this way, the second region blocks of multiple chromosomes are obtained according to steps (2)-(4).

[0109] (6) Screen the second region blocks, and select the second region blocks with a population frequency of 0.3-0.7 as the linkage SNP region blocks.

[0110] Preferably, in step (2), in order to reduce the amount of calculation, the LD values of only the SNP sites with abs(AF-0.5) less than a fourth threshold T4 can be calculated, and the T4 is 0.01-0.04.

[0111] Among them, only the second region blocks with LD values less than 0.1 between the second region blocks include: sorting the second region blocks according to the number of linkage sites from large to small, calculating the LD values between the current second region block and the previous and retained second region blocks from the second second region block, if all the LD values are greater than 0.1, the current second region block is retained as a linkage SNP region block; the calculation process of the LD values between the second region blocks is: taking any one of the linkage sites in the second region block to calculate.

[0112] Among them, for the population frequency of the second region block, the region block 1 is the highest frequency in all region blocks, so the frequency of the region block 1 is basically above 0.5, and the region block 2 is the opposite combination corresponding to each SNP position of the region block 1. The linkage SNP region blocks on chromosome 1 are selected, as shown in Table 2.

[0113] Table 2 Selected linkage SNP region block on chromosome 1

[0114] chromosome SNP start position haplotype 1 haplotype 2 frequency of haplotype 1 chr1 924310 1111111111 ·0000000000 0.558 chr1 1188877 11111111111111 ·00000000000000 0.571 chr1 1651785 ·00000000000000 11111111111111 0.584 chr1 1935938 1111111111 ·0000000000 0.642 chr1 1982826 ·000000000000 111111111111 0.651 chr1 2085406 11111111111 ·00000000000 0.665 chr1 2113103 1111111111 ·0000000000 0.656 chr1 2866744 1111110111 ·0000001000 0.524 chr1 2924136 111111111111 ·000000000000 0.594

[0115] wherein, in units of linkage SNP region block: aligning the linkage SNP region block as a whole with the reference genome, typing according to the sequencing depth ratio, the sequencing depth of the linkage SNP region block is the superposition of the sequencing depth of the corresponding typed linkage site within it, which can equivalently increase the sequencing depth.

[0116] Specifically, in step S1, the samples are the pregnant woman's free sample S, the maternal leukocyte sample M, and the to-be-tested paternal sample F. The pregnant woman's free blood, maternal leukocytes, and to-be-tested father are subjected to conventional WGS experiments. The sequencing depth of the pregnant woman's free blood sample S, the pregnant woman's leukocyte sample M, and the to-be-tested suspected father sample F is required to be greater than 3x, 3x, and 2x, respectively. The sequencing depth can be appropriately reduced for high gestational weeks and high fetal concentration. The sequencing data is filtered according to the conventional method.

[0117] In step S3, according to the typing of the linkage SNP region block and the population frequency, the genotype of the fetus is obtained by the sequencing depth ratio of the linkage SNP region block of S and M, and the genotype of the father is obtained by the sequencing depth ratio of the linkage SNP region block of F; the paternity is identified by calculating the error rate and CPI. After data filtering, each SNP linkage region block site of each sample has a sequencing depth, and a "wild type" and "mutant" site depth determined according to the human genome reference sequence. Taking a certain region block site as an example, A represents a wild type site, and a represents a mutant site. If the total depth of the site (referring to the SNP linkage region block, the same below) in the sequencing result is 100X, of which A is 100X and a is 0X, then the site is a homozygous wild type site; if A is 0X and a is 100X, then it is a homozygous mutant site; two homozygous sites are denoted as homozygotes; if the sequencing depths of A and a are close to 1:1, then the site is denoted as a heterozygote.

[0118] The typing of the fetus, specifically:

[0119] 1) When the sequencing depth ratio of S at the site is 0.01

[0120] 2) S in the site of the sequencing depth ratio is 0.01

[0121] In the process of identification, the end point value of the sequencing depth ratio can also be adjusted according to the quality, accuracy and the like of the sample.

[0122] F sample typing:

[0123] Theoretically, the regional block distribution of the F sample should be composed of homozygotes and heterozygotes. Considering the partial pollution introduced in the sampling and transportation process, the F will produce a heterologous signal similar to the fetal information site. When typing F, the heterologous signal is removed in the following method.

[0124] When the sequencing depth ratio of F at the site is A / (A+a) < 0.16, the genotype of F is considered to be aa.

[0125] When the sequencing depth ratio of F at the site is a / (A+a) < 0.16, the genotype of F is considered to be AA.

[0126] In other cases, the genotype of F is considered to be Aa.

[0127] In the process of identification, the end point value of the sequencing depth ratio can also be adjusted according to the quality, accuracy and the like of the sample.

[0128] The present application is described by taking triplet paternity identification method as an example:

[0129] According to Mendelian inheritance law, when it is determined that the genotype of the pregnant woman herself is aa, and the genotype of the fetus contains one A, if the suspect father F can provide A at this site, it represents that the site meets the Mendelian inheritance law and is a matching site. Otherwise, it is a mismatch site. Based on this principle, the mismatch rate between the fetus information site and the suspect father F and the PI value of each corresponding site are calculated, and finally the CPI is obtained. The calculation of PI and CPI refers to the calculation method of triplet in the national standard | GB / T 37223-2018. According to the standard, when CPI > 10000 (CPI logarithm > 4), it supports that the father to be tested and the fetus are in parent-child relationship, and the theoretical mismatch rate is 0; when CPI < 0.0001 (CPI logarithm < -4), it is excluded that the father to be tested and the fetus are in parent-child relationship, and the theoretical mismatch rate is much greater than 0. The method for paternity identification according to PI and CPI is well known to those skilled in the art.

[0130] Example 1: Acquisition of mapping relationship between point site and linkage SNP regional block

[0131] The embodiment discloses a method for acquiring mapping relationship of point sites and linkage SNP region blocks, and the process is as follows:

[0132] (1) Take 718 autosomal WGS data of CHS, CHB and CDX three populations in 1KGP (The 1000 Genomes Project, 1000G / 1KGP), and take SNP with a population frequency of 0.2-0.8. Remove indel type sites, remove SNP sites related to STR, remove SNP sites related to MSI, remove regions with possible duplication, and remove regions with high GC content and low complexity near SNP.

[0133] (2) On a chromosome, calculate from front to back, fix the first SNP as SNP1, calculate the LD value between the subsequent SNP site (SNP site with abs(AF-0.5) less than 0.02) and SNP1, and count these values until at least 10 LD values in the first 12 LD values are less than 0.1, and SNP1 to the cutoff site is the first region block.

[0134] The calculation process of the LD value is consistent with the prior art. The LD value calculation method is shown as follows; for example, the SNP genotype at a certain position is T, and the allele genotype is C; and the SNP genotype at a certain position connected thereto is T, and the allele genotype is C. Then there are four region blocks of TT, TC, CT and CC in the population, sum_ is recorded as sum([TT, TC, CT, CC]), PT1 is (TT+TC) / sum_, PT2 is (TT+CT) / sum_, D is (TT / sum_)-PT1*PT2, and LD is D*D / (PT1*PT2*(1-PT1)*(1-PT2)). For example, the LD value of the site at position 213204 of chr4 is shown in the following table. Figure 2 The horizontal coordinate is the base position, and the vertical coordinate is the LD value. Figure 2 The following table is the mapping relationship diagram of the base position and the LD value of the site at position 213204 in the method of the embodiment.

[0135] (3) The SNP sites in the first region block are screened, the SNP sites with an LD value greater than 0.95 are taken as linkage sites, and the linkage sites in the first region block form the second region block. The region block corresponding to the second region block is judged, if P(hap1)+P(hap2)>0.98, the second region block is retained as a linkage SNP region block; otherwise, the second region block is deleted.

[0136] (4) In this way, the subsequent adjacent linkage sites are fixed in turn, and the LD value is calculated with the subsequent SNP sites, and the second region block on the chromosome is obtained according to steps (2) and (3); the second region blocks are sorted according to the number of linkage sites from large to small, and the LD value between the current second region block and the previous and retained second region block is calculated from the second second region block; if all the LD values are greater than 0.1, the current second region block is retained as a linkage SNP region block; the calculation process of the LD value between the second region blocks is as follows: any one linkage site in the second region block is taken to calculate.

[0137] (5) In this way, the second region blocks of 22 chromosomes are obtained according to steps (2)-(4).

[0138] (6) The second region blocks are screened, and the second region blocks with a population frequency of 0.3-0.7 are selected as linkage SNP region blocks, and the mapping relationship between the point sites and the linkage SNP region blocks is presented in the form of a database, such as forming a hap database.

[0139] Example 2: Non-invasive prenatal paternity identification

[0140] This example experiments 3 groups of samples, corresponding to S1-S3 families, and the corresponding pregnant women peripheral blood free samples, maternal leukocytes and the father to be tested, which are marked as S1-S3, M1-M3 and F1-F3. The control example is experimentally obtained by using the method of liquid phase hybridization capture and high-throughput sequencing, and the fetal concentration is distributed in 1%-3%, and it is determined that the 3 groups of fetuses and the father to be tested F are in parent-child relationship. The 3 groups of samples are subjected to conventional WGS experiment, sequencing data filtering, sequencing depth is obtained, and region block typing and concentration evaluation are performed, and the results are shown in Table 3.

[0141] Table 3: Region block sequencing depth and concentration evaluation

[0142]

[0143]

[0144] The mismatch rate and CPI between the 3 groups of samples are statistically analyzed, and the parent-child relationship is determined according to the results. The results are shown in Table 4:

[0145] Table 4: Parent-child relationship determination

[0146]

[0147] In Table 4, the WGS conclusion represents the conclusion determined according to the method of the present patent, and the NGS conclusion represents the conclusion determined by using the existing hybridization method.

[0148] As can be seen from Table 3, the sequencing depth of the application is 7-11x, which is low, and the sample meets the requirements. As can be seen from Table 4, the method of the application is consistent with the existing hybridization method. It can be seen that, by optimizing the algorithm and analyzing the linkage SNP region block, the application can accurately determine the parent-child relationship in low-depth WGS data, which not only compresses the experimental period and can obtain the conclusion within 24h, but also maintains high accuracy.

[0149] Example 3 Mapping relationship between fetal information point number and CPI value

[0150] In practical application, the fetal information point number is related to the sequencing depth and the fetal concentration. In order to determine how many fetal information point numbers are needed to accurately obtain the parentage identification result, i.e. the CPI logarithmic value is greater than 4 or less than -4, the S sample information point number is randomly sampled, and the corresponding F is compared to calculate the CPI value, and the result is shown in Figure 3

[0151] The sampling rules are as follows: the information points are sampled from 100 to 610, with an interval of 30 points each time. If the information point number is not sampled, according to the real data, 6 times of random sampling are repeated each time, and the CPI average value is taken. The corresponding curve graph of the information point number and the CPI is obtained. It can be known that when the information point number is only 100, the CPI logarithmic value of the family meeting the parent-child relationship is much greater than 4, and the CPI logarithmic value of the combination not meeting the parent-child relationship is much less than -4. When actually applied, it can be specified that the fetal information point number is at least greater than 100, and then the parentage result identification can be performed.

[0152] The above embodiments of the present disclosure are only examples for clearly illustrating the present disclosure, and are not a limitation on the embodiments of the present disclosure. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments are not required to be exhausted. Any modification, equivalent replacement and improvement made within the spirit and principle of the present disclosure shall be included in the protection scope of the claims of the present disclosure.​

Claims

1. A method for rapid parentage testing by linkage of blocks of loci with polymorphisms, characterized in that, The method comprises the following steps: S1: establishing a linkage relationship database of polymorphic sites; S2: performing low-depth WGS sequencing on samples according to the requirements of parentage identification to obtain sequencing data; S3: screening, according to the linkage relationship of the polymorphic sites, typing data of corresponding sites constituting a linkage polymorphic site region block on a reference genome in the sequencing data; S4: performing parentage identification according to the typing data and in units of linkage polymorphic site region blocks; Wherein, the linkage polymorphic site region block is a linkage polymorphic site region block composed of a plurality of linkage sites with high linkage degree, and the reference genome is public genomic WGS data corresponding to a population or a population; The unit of the linkage polymorphic site region block is: aligning the linkage SNP region block as a whole with the reference genome, typing according to the sequencing depth ratio, and the sequencing depth of the linkage SNP region block is the superposition of the sequencing depth of the corresponding typed linkage sites therein; Wherein, the polymorphic site is SNP.

2. The method of claim 1, wherein, In step S1, the linkage relationship of the polymorphic sites is obtained by at least one of the following ways: (1) obtained by processing public genomic WGS data; (2) obtained by processing a large amount of high-depth WGS measured data; (3) obtained from haplotype cell sequencing data; (4) obtained by processing long sequencing mode data; (5) obtained by querying public databases or literature.

3. The method of claim 2, wherein, Wherein, the public genomic WGS data processed or the large amount of high-depth WGS measured data processed is specifically screened based on the reference genome according to a predetermined rule.

4. The method of claim 3, wherein, The predetermined rule is: (1) from G WGS data of autosomes of the reference genome, take the SNP with a population frequency of 0.2-0.8, and remove sites and regions that do not meet the requirements; (2) on a chromosome, calculate from front to back, fix the first SNP site as SNP1, calculate the LD value, i.e. linkage disequilibrium value, between the subsequent SNP sites and SNP1, and count these values until at least K LD values in the first M LD values are less than the first threshold T1, and SNP1 to the cutoff site is the first region block; (3) screen the SNP sites in the first region block, take the SNP sites with LD values greater than the second threshold T2 as linkage sites, and the linkage sites in the first region block form the second region block; then judge the region block corresponding to the second region block, if P(hap1)+P(hap2)>T3, the second region block is retained; otherwise, the second region block is deleted; wherein, hap1 and hap2 are the two most frequent different region blocks in the second region, P(hap1) is the frequency of region block hap1 appearing in G data, P(hap2) is the frequency of region block hap2 appearing in G data, and T3 is the third threshold. (4) Similarly, subsequently fix the next adjacent linkage site, and then calculate the LD value with the subsequent SNP site in turn, to obtain all the second region blocks on the chromosome according to the steps (2)-(3); sort all the second region blocks according to the number of linkage sites, and only keep the second region blocks with the LD value between the second region blocks being less than a fourth threshold T4; (5) Similarly, obtain the second region blocks of multiple chromosomes according to the steps (2)-(4); (6) Screen the second region blocks of the multiple chromosomes, and select the second region blocks with the population frequency being between 0.2-0.8 as the linkage SNP region blocks.

5. The method of claim 4, wherein, The G is greater than 200.

6. The method of claim 3, wherein, The predetermined rule is: obtain the region blocks of gametes of a specific population: select the semen of 400-1000 individuals in the population, separate and obtain single sperm, perform single-cell second-generation sequencing to obtain sequencing data; align the sequencing data to the reference genome to obtain the data of 400-1000 haplotypes; obtain the frequency data of independent polymorphisms of the population by aligning the data of the 400-1000 haplotypes; the germ cell haplotype is a kind of specialization for describing the linkage region blocks, and the SNPs with the population frequency being between 0.2-0.8 are taken and the sites and regions that do not meet the requirements are removed; expand and connect all SNPs within 100 kbp from each SNP above as the linkage group of each SNP, and screen the SNP linkage group by the following conditions: the sum of the frequencies of a specific region block P and its allodifferent region block Q in the linkage group is greater than 0.95, and the region block P covers more than N SNPs, wherein N is an integer greater than or equal to 2; the specific region block P includes a missing region block; if a certain region block is AAAaaa, then aaaAAA is its allodifferent region block, and A-Aaaa and AAA--a are its missing region blocks.

7. The method of claim 3, wherein, The predetermined rule is: select 400-1000 individuals in the population, obtain the haplotype sequencing data of the whole genome range with a fragment length of >50 kb by third-generation sequencing; align the sequencing data to the reference genome to obtain the data of 400-1000 haplotypes; obtain the frequency data of independent polymorphisms of the population by aligning the data of the 400-1000 haplotypes; the long sequencing haplotype is a kind of specialization for describing the linkage region blocks, and the SNPs with the population frequency being between 0.2-0.8 are taken and the sites and regions that do not meet the requirements are removed; expand and connect all SNPs within 100 kbp from each SNP above as the linkage group of the SNP, and screen the SNP linkage group by the following conditions: the sum of the frequencies of a specific region block P and its allodifferent region block Q in the linkage group is greater than 0.95, and the region block P covers more than N SNPs, wherein N is an integer greater than or equal to 2; the specific region block P includes a missing region block; if a certain region block is AAAaaa, then aaaAAA is its allodifferent region block, and A-Aaaa and AAA--a are its missing region blocks.

8. The method of claim 3, wherein, The predetermined rule is: using the public database LDLink, screening the specific population's regional block by LDhap of LDLinkR software package, taking the SNP of Asian population in the database between 0.2-0.8 and removing the sites and regions that do not meet the requirements; taking each SNP above as a starting point, expanding all SNPs within 100kbp connected to the SNP, and screening the SNP linkage group by the following conditions: the sum of the frequencies of a certain specific regional block P and its alien regional block Q in the linkage group is greater than 0.95, and the regional block P covers more than N SNPs, wherein N is an integer greater than or equal to 2; the specific regional block P includes missing regional blocks; if a certain regional block is AAAaaa, then aaaAAA is its alien regional block, and A-Aaaa and AAA--a are its missing regional blocks.

9. The method according to any one of claims 1 to 8, characterized in that, In the step S1, the sample is a pregnant woman's free sample S, a maternal leukocyte sample M and a sample F to be tested for the father; in the step S3, according to the typing of the linkage SNP regional block and the population frequency, the genotype of the fetus is obtained by the sequencing depth ratio of the linkage SNP regional block of S and M, and the genotype of the father is obtained by the sequencing depth ratio of the linkage SNP regional block of F; the parentage identification is carried out by calculating the error rate and CPI.

10. The method of claim 9, wherein, The process of obtaining the genotype of the fetus is: if the sequencing depth ratio of S at the site is 0.01 11. The method according to any one of claims 1 to 8, characterized in that, The parentage identification is selected from the father-child relationship, mother-child relationship, sibling relationship, half-sibling relationship, uncle-niece relationship, grandparent-grandchild relationship or great-grandparent-great-grandchild relationship; the parentage identification is selected from non-invasive prenatal parentage identification.

12. The method according to any one of claims 1 to 8, characterized in that, The method is suitable for diploid organisms that meet Mendelian inheritance.

13. The method of claim 12, wherein, The method is suitable for humans. The predetermined rule is: using the public database LDLink, screening the specific population's regional block by LDhap of LDLinkR software package, taking the SNP of Asian population in the database between 0.2-0.8 and removing the sites and regions that do not meet the requirements; taking each SNP above as a starting point, expanding all SNPs within 100kbp connected to the SNP, and screening the SNP linkage group by the following conditions: the sum of the frequencies of a certain specific regional block P and its alien regional block Q in the linkage group is greater than 0.95, and the regional block P covers more than N SNPs, wherein N is an integer greater than or equal to 2; the specific regional block P includes missing regional blocks; if a certain regional block is AAAaaa, then aaaAAA is its alien regional block, and A-Aaaa and AAA--a are its missing regional blocks. In the step S1, the sample is a pregnant woman's free sample S, a maternal leukocyte sample M and a sample F to be tested for the father; in the step S3, according to the typing of the linkage SNP regional block and the population frequency, the genotype of the fetus is obtained by the sequencing depth ratio of the linkage SNP regional block of S and M, and the genotype of the father is obtained by the sequencing depth ratio of the linkage SNP regional block of F; the parentage identification is carried out by calculating the error rate and CPI. The process of obtaining the genotype of the fetus is: if the sequencing depth ratio of S at the site is 0.01 The parentage identification is selected from the father-child relationship, mother-child relationship, sibling relationship, half-sibling relationship, uncle-niece relationship, grandparent-grandchild relationship or great-grandparent-great-grandchild relationship; the parentage identification is selected from non-invasive prenatal parentage identification. The method is suitable for diploid organisms that meet Mendelian inheritance. The method is suitable for humans.

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