A high-throughput sequencing heterozygosity reduction risk annotation method and system

Abstract

The present invention discloses a high-throughput sequencing heterozygosity reduction risk annotation method, which performs heterozygosity reduction risk screening on the proband loci of three-sample high-throughput sequencing data of a family and marks the genetic source, including marking the risk of the proband homozygous loci and marking the genetic source of the loci at the same time. The specific screening and marking are as follows: screening and marking new variants with reliable sequencing quality and homozygous population frequency, that is, screening and marking variants that neither homozygous parent has; screening and marking variants that are only inherited from the father with reliable sequencing quality and homozygous population frequency; screening and marking variants that are only inherited from the mother with reliable sequencing quality and homozygous population frequency; screening and marking variants that both parents have with reliable sequencing quality and homozygous population frequency. The present invention also discloses a high-throughput sequencing heterozygosity reduction risk annotation system, which has high analysis efficiency, clear logic and high detection rate.

Description

Technical Field

[0001] The present invention relates to the technical field of data analysis, and in particular to a high-throughput sequencing heterozygosity reduction risk annotation method and system. Background Art

[0002] Human chromosomes are composed of 23 pairs of autosomes and 2 sex chromosomes. For sex chromosomes, males are XY and females are XX. Under normal circumstances, one of the paired chromosomes comes from the father and the other comes from the mother, which are called homologous chromosomes.

[0003] Chromosomes are linear structures formed by DNA wrapped around histones. High-throughput sequencing technology can be used to sequence the DNA on chromosomes and obtain DNA sequences. Among them, whole exome sequencing is a commonly used high-throughput sequencing technology, which can detect about 20,000 genes in the genome. Some of the detected gene variants are homozygous, that is, the alleles at the same site on homologous chromosomes are the same, and some are heterozygous, that is, the alleles at the same site on homologous chromosomes are the same. Heterozygosity refers to the frequency of alleles at a certain locus being heterozygous. Under normal circumstances, variants are inherited from parents respectively and appear randomly. The heterozygosity is relatively constant. When homozygosity appears continuously, it indicates that there may be a phenomenon of reduced heterozygosity. Among them, when the homozygous variant is inherited from only one parent, that is, only one parent has the variant at this site, it indicates that there may be a large fragment of heterozygous deletion or uniparental diploidy, which further leads to reduced heterozygosity. Reduced heterozygosity is one of the important causes of genetic diseases. At present, there is no method and system for annotating sequencing data for direct risk warnings. Summary of the invention

[0004] The object of the present invention is to solve at least one of the technical drawbacks.

[0005] To this end, the purpose of the present invention is to provide a high-throughput sequencing heterozygosity reduction risk annotation method and system.

[0006] In order to achieve the above-mentioned purpose, an embodiment of the present invention provides a high-throughput sequencing heterozygosity reduction risk annotation method, which performs heterozygosity reduction risk screening on the proband loci of the three-sample high-throughput sequencing data of the family and marks the genetic source, including risk marking of the homozygous loci of the proband and simultaneous screening of the genetic source of the loci; the simultaneous screening of the genetic source of the loci is specifically as follows: screening and marking new variants with reliable sequencing quality and homozygous population frequency, that is, screening and marking variants that neither homozygous parent has; screening and marking variants that are only inherited from the father with reliable sequencing quality and homozygous population frequency; screening and marking variants that are only inherited from the mother with reliable sequencing quality and homozygous population frequency; screening and marking variants that both parents have with reliable sequencing quality and homozygous population frequency.

[0007] Preferably, the sequencing quality is reliable, specifically the ratio of the total amount of bases obtained by sequencing to the genome size is not less than 25X.

[0008] In any of the above schemes, it is preferred that if a homozygous variation inherited only from the father or a homozygous variation inherited only from the mother and a variation marker appear continuously, it indicates a heterozygous deletion of a long segment or an entire chromosome or a uniparental inherited segment or uniparental diploidy, and thus indicates a reduced heterozygosity.

[0009] In any of the above schemes, it is preferred that the newly emerged variants with reliable sequencing quality and homozygous population frequency are screened and marked, specifically:

[0010] Set S1 conditions: the heterozygous or homozygous column of the proband's variant site is equal to homozygous, the population frequency of the public database is less than or equal to 0.05, the coverage depth of the proband's variant site is equal to or greater than 25, the frequency of the local database is less than 0.05, the filter label of the proband's GATK is equal to PASS, the heterozygous or homozygous column of the father's variant site is empty, and the heterozygous or homozygous column of the mother's variant site is empty.

[0011] Variants that meet the S1 criteria are screened, and the selected variants are marked as new variants with reliable sequencing quality and homozygous population frequency.

[0012] In any of the above schemes, it is preferred that the variants inherited only from the father with reliable sequencing quality and homozygous population frequency are screened for markers, specifically:

[0013] Set S2 conditions: the heterozygous or homozygous column of the proband's variant site is equal to homozygous, the population frequency of the public database is less than or equal to 0.05, the coverage depth of the proband's variant site is equal to or greater than 25, the frequency of the local database is less than 0.05, the filter label of the proband's GATK is equal to PASS, the heterozygous or homozygous column of the father's variant site is not a null value, and the heterozygous or homozygous column of the mother's variant site is a null value.

[0014] Variants that meet the S2 criteria are screened, and the selected variants are marked as variants that are only inherited from the father with reliable sequencing quality and homozygous population frequency.

[0015] In any of the above schemes, it is preferred that the variants inherited only from the mother with reliable sequencing quality and homozygous population frequency are screened for markers, specifically:

[0016] Set S3 conditions: the heterozygous or homozygous column of the proband's variant site is equal to homozygous, the population frequency of the public database is less than or equal to 0.05, the coverage depth of the proband's variant site is equal to or greater than 25, the frequency of the local database is less than 0.05, the filter label of the proband's GATK is equal to PASS, the heterozygous or homozygous column of the father's variant site is empty, and the heterozygous or homozygous column of the mother's variant site is not empty.

[0017] Variants that meet the S3 criteria are screened, and the selected variants are marked as variants that are only inherited from the mother with reliable sequencing quality and homozygous population frequency.

[0018] In any of the above schemes, it is preferred to screen markers for variants that are of reliable sequencing quality and homozygous in the population frequency and that are shared by both parents, specifically:

[0019] Set S4 conditions: the heterozygous or homozygous column of the proband's variant site is equal to homozygous, the population frequency of the public database is less than or equal to 0.05, the coverage depth of the proband's variant site is equal to or greater than 25, the frequency of the local database is less than 0.05, the filter label of the proband's GATK is equal to PASS, the heterozygous or homozygous column of the father's variant site is not a null value, and the heterozygous or homozygous column of the mother's variant site is not a null value.

[0020] Variants that meet the S4 criteria are screened, and the selected variants are marked as variants that have reliable sequencing quality and homozygous population frequency and are present in both parents.

[0021] In any of the above schemes, it is preferred that the screening of population frequency is set to be less than 0.05.

[0022] The present invention also proposes a high-throughput sequencing heterozygosity reduction risk annotation system, which is used to screen the proband's sites for heterozygosity reduction risk and mark the genetic source of the three-sample high-throughput sequencing data of the family, including: marking the risk of the homozygous sites of the proband and simultaneously screening the genetic source of the site, wherein the high-throughput sequencing heterozygosity reduction risk annotation system includes: a first screening marker module, a second screening marker module, a third screening marker module and a fourth screening marker module, and the screening and marking are:

[0023] The first screening marker module performs screening markers for new variants with reliable sequencing quality and homozygous population frequency, that is, screening markers for variants that neither parent has.

[0024] The second screening marker module performs screening of variant markers that are inherited only from the father and have reliable sequencing quality and homozygous population frequency.

[0025] The third screening marker module performs screening markers for variants that are only inherited from the mother and have reliable sequencing quality and homozygous population frequency.

[0026] The fourth screening marker module performs screening of variant markers that are shared by both parents with reliable sequencing quality and homozygous population frequency.

[0027] Preferably, the sequencing quality is reliable, specifically the ratio of the total amount of bases obtained by sequencing to the genome size is not less than 25X.

[0028] In any of the above schemes, it is preferred that when homozygous variants inherited only from the father or homozygous variants inherited only from the mother appear continuously with the variant markers, it indicates a heterozygous deletion of a long segment or an entire chromosome or a uniparental inherited segment or uniparental diploidy, and thus indicates a reduction in heterozygosity.

[0029] In any of the above schemes, preferably, the first marker screening module performs screening of markers for emerging variants with reliable sequencing quality and homozygous population frequency, specifically:

[0030] Set S1 conditions: the heterozygous or homozygous column of the proband's variant site is equal to homozygous, the population frequency of the public database is less than or equal to 0.05, the coverage depth of the proband's variant site is equal to or greater than 25, the frequency of the local database is less than 0.05, the filter label of the proband's GATK is equal to PASS, the heterozygous or homozygous column of the father's variant site is empty, and the heterozygous or homozygous column of the mother's variant site is empty.

[0031] Variants that meet the S1 criteria are screened, and the selected variants are marked as new variants with reliable sequencing quality and homozygous population frequency.

[0032] In any of the above schemes, preferably, the second screening marker module performs screening markers on the variants inherited only from the father with reliable sequencing quality and homozygous population frequency, specifically:

[0033] Set S2 conditions: the heterozygous or homozygous column of the proband's variant site is equal to homozygous, the population frequency of the public database is less than or equal to 0.05, the coverage depth of the proband's variant site is equal to or greater than 25, the frequency of the local database is less than 0.05, the filter label of the proband's GATK is equal to PASS, the heterozygous or homozygous column of the father's variant site is not a null value, and the heterozygous or homozygous column of the mother's variant site is a null value.

[0034] Variants that meet the S2 criteria are screened, and the selected variants are marked as variants that are only inherited from the father with reliable sequencing quality and homozygous population frequency.

[0035] In any of the above schemes, preferably, the third screening marker module performs screening markers on the variants that are only inherited from the mother and have reliable sequencing quality and homozygous population frequency, specifically:

[0036] Set S3 conditions: the heterozygous or homozygous column of the proband's variant site is equal to homozygous, the population frequency of the public database is less than or equal to 0.05, the coverage depth of the proband's variant site is equal to or greater than 25, the frequency of the local database is less than 0.05, the filter label of the proband's GATK is equal to PASS, the heterozygous or homozygous column of the father's variant site is empty, and the heterozygous or homozygous column of the mother's variant site is not empty.

[0037] Variants that meet the S3 criteria are screened, and the selected variants are marked as variants that are only inherited from the mother with reliable sequencing quality and homozygous population frequency.

[0038] In any of the above schemes, preferably, the fourth screening marker module performs screening markers on variants that are both present in parents with reliable sequencing quality and homozygous population frequency, specifically:

[0039] Set S4 conditions: the heterozygous or homozygous column of the proband's variant site is equal to homozygous, the population frequency of the public database is less than or equal to 0.05, the coverage depth of the proband's variant site is equal to or greater than 25, the frequency of the local database is less than 0.05, the filter label of the proband's GATK is equal to PASS, the heterozygous or homozygous column of the father's variant site is not a null value, and the heterozygous or homozygous column of the mother's variant site is not a null value.

[0040] Variants that meet the S4 criteria are screened, and the selected variants are marked as variants that have reliable sequencing quality and homozygous population frequency and are present in both parents.

[0041] In any of the above schemes, preferably, the screening condition of population frequency is set to be less than 0.05.

[0042] According to the high-throughput sequencing heterozygosity reduction risk annotation method and system of the embodiment of the present invention, the high-throughput sequencing data is directly marked for the risk of heterozygosity reduction and the corresponding sites are marked for the genetic source, so that the analysis of heterozygosity reduction is intuitive and the analysis efficiency is improved. The present invention screens the sites of reduced heterozygosity and marks the sources of variation of three samples in the family. The marking items are few and the logic is clear. The true positive results of the pathogenic causes of reduced heterozygosity can be quickly identified, and the low-risk samples can be easily predicted and missed, which provides convenience for the analysis of the pathogenic causes of reduced heterozygosity, enriches the analysis method of high-throughput annotation data, and improves the detection rate.

[0043] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0045] Figure 1 A flowchart of a method for reducing risk annotation of heterozygosity in high-throughput sequencing according to an embodiment of the present invention;

[0046] Figure 2 4 is a structural diagram of a high-throughput sequencing heterozygosity risk reduction annotation system according to an embodiment of the present invention. DETAILED DESCRIPTION

[0047] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0048] The present invention provides a high-throughput sequencing heterozygosity reduction risk annotation method, which uses three-family sample combined data screening to mark variants indicating reduced heterozygosity.

[0049] like Figure 1 As shown, a high-throughput sequencing heterozygosity reduction risk annotation method is used to screen the proband's loci for heterozygosity reduction risk and mark the genetic source of the proband's three-sample high-throughput sequencing data, including risk marker 1 for the proband's homozygous loci and simultaneous screening marker 2 for the genetic source of the loci; the simultaneous screening markers for the genetic source of the loci are specifically: screening markers for new variants with reliable sequencing quality and homozygous population frequency, that is, screening marker 3 for variants that neither homozygous parent has; screening marker 4 for variants that are only inherited from the father with reliable sequencing quality and homozygous population frequency; screening marker 5 for variants that are only inherited from the mother with reliable sequencing quality and homozygous population frequency; screening marker 6 for variants that both parents have with reliable sequencing quality and homozygous population frequency.

[0050] Furthermore, the reliable sequencing quality means that the ratio of the total number of bases obtained by sequencing to the genome size is not less than 25X.

[0051] Furthermore, if a homozygous variant inherited only from the father or a homozygous variant inherited only from the mother and a variant marker appear consecutively, it indicates a heterozygous deletion of a long segment or an entire chromosome or a uniparental inherited segment or uniparental diploidy, which in turn indicates a decrease in heterozygosity.

[0052] Furthermore, new variants with reliable sequencing quality and homozygous population frequency were screened for markers (abbreviated as LOH_N), specifically:

[0053] Set S1 conditions: the heterozygous or homozygous column of the proband's variant site (abbreviated as Het / Hom_proband) is equal to homozygous (abbreviated as Hom), the population frequency of the public database (abbreviated as fmax) is less than or equal to 0.05, the coverage depth of the proband's variant site (abbreviated as Alt-Cover_proband) is equal to or greater than 25, the local database frequency (abbreviated as local) is less than 0.05, the filter label of the proband's GATK (abbreviated as Filter_proband) is equal to PASS, the heterozygous or homozygous column of the father's variant site (abbreviated as Het / Hom_father) is empty, and the heterozygous or homozygous column of the mother's variant site (abbreviated as Het / Hom_mother) is empty.

[0054] Variants that meet the S1 criteria are screened, and the selected variants are marked as new variants with reliable sequencing quality and homozygous population frequency.

[0055] Screening markers (abbreviated as LOH_Pat) are performed for variants that are only inherited from the father and have reliable sequencing quality and homozygous population frequency. Specifically:

[0056] Set S2 conditions: the heterozygous or homozygous column of the proband's variant site (abbreviated as Het / Hom_proband) is equal to homozygous (abbreviated as Hom), the population frequency of the public database (abbreviated as fmax) is less than or equal to 0.05, the coverage depth of the proband's variant site (abbreviated as Alt-Cover_proband) value is equal to or greater than 25, the local database frequency (abbreviated as local) is less than 0.05, the filter label of the proband's GATK (abbreviated as Filter_proband) is equal to PASS, the heterozygous or homozygous column of the father's variant site (abbreviated as Het / Hom_father) is not a null value, and the heterozygous or homozygous column of the mother's variant site (abbreviated as Het / Hom_mother) is a null value.

[0057] Variants that meet the S2 criteria are screened, and the selected variants are marked as variants that are only inherited from the father with reliable sequencing quality and homozygous population frequency.

[0058] Screening markers (abbreviated as LOH_Mat) for variants that are only inherited from the mother and have reliable sequencing quality and homozygous population frequency, specifically:

[0059] Set S3 conditions: the proband's variant site heterozygous or homozygous column (abbreviated as Het / Hom_proband) is equal to homozygous (abbreviated as Hom), the public database population frequency (abbreviated as fmax) is less than or equal to 0.05, the proband's variant site coverage depth (abbreviated as Alt-Cover_proband) is equal to or greater than 25, the local database frequency (abbreviated as local) is less than 0.05, the proband's GATK filter label (abbreviated as Filter_proband) is equal to PASS, the father's variant site heterozygous or homozygous column (abbreviated as Het / Hom_father) is a null value, and the mother's variant site heterozygous or homozygous column (abbreviated as Het / Hom_mother) is not a null value.

[0060] Variants that meet the S3 criteria are screened, and the selected variants are marked as variants that are only inherited from the mother with reliable sequencing quality and homozygous population frequency.

[0061] Screening markers (abbreviated as LOH_Pat_Mat) are performed for variants that are of reliable sequencing quality and homozygous in the population frequency and that are shared by both parents. Specifically:

[0062] Set S4 conditions: the proband's variant site heterozygous or homozygous column (abbreviated as Het / Hom_proband) is equal to homozygous (abbreviated as Hom), the public database population frequency (abbreviated as fmax) is less than or equal to 0.05, the proband's variant site coverage depth (abbreviated as Alt-Cover_proband) is equal to or greater than 25, the local database frequency (abbreviated as local) is less than 0.05, the proband's GATK filter label (abbreviated as Filter_proband) is equal to PASS, the father's variant site heterozygous or homozygous column (abbreviated as Het / Hom_father) is not a null value, and the mother's variant site heterozygous or homozygous column (abbreviated as Het / Hom_mother) is not a null value.

[0063] Variants that meet the S4 criteria are screened, and the selected variants are marked as variants that have reliable sequencing quality and homozygous population frequency and are present in both parents.

[0064] Specifically, the screening condition of population frequency was set to be less than 0.05. If the population frequency was less than 0.05, it was considered that the variant supported benign evidence.

[0065] Screening marker description: The markers LOH_N and LOH_Pat_Mat that appear at intervals are of little significance. When they appear consecutively with LOH_Pat or LOH_Mat markers and mutations, they indicate heterozygous deletion of long segments or whole chromosomes or uniparental inheritance segments or uniparental diploidy, which in turn indicates reduced heterozygosity. In order to increase the true positive rate, the screening setting of the population frequency is preferably less than 0.05, and a population frequency less than this is considered to be evidence that the variant supports benignity. Specific embodiment:

[0067] An example of whole exome sequencing data of three samples from a family in our laboratory. The number of detected variant sites is 83,860. The identifier and header description of the first line of bioinformatics annotation, i.e., the header, are shown in Table 1 below:

[0068]

[0069] Table 1

[0070] The screening and analysis results are shown in Table 2 below:

[0071]

[0072] Table 2

[0073] If the data is not screened according to the scheme of the present invention, but the homozygous loci of the proband are directly screened, the results shown in the following Table 3 will be obtained:

[0074]

[0075] Table 3

[0076] Compared with the control screening method, the patent scheme screens and marks the homozygous sites for risk, with a small number of markers (only 12) and strong analyzability. The above embodiment quickly identifies possible clinical pathogenic variants. The control group screened 22,626 markers, which could not distinguish the risk of reduced heterozygosity, and therefore could not analyze possible clinical pathogenic variants.

[0077] like Figure 2 As shown, the present invention also proposes a high-throughput sequencing heterozygosity reduction risk annotation system, which is used to screen the proband's sites for heterozygosity reduction risk and mark the genetic source of the three-sample high-throughput sequencing data of the family, including: marking the risk of the homozygous sites of the proband and simultaneously screening the genetic source of the site, wherein the high-throughput sequencing heterozygosity reduction risk annotation system includes: a first screening marker module 100, a second screening marker module 200, a third screening marker module 300 and a fourth screening marker module 400, and the screening and marking are:

[0078] The first screening marker module 100 performs screening for novel variants (abbreviated as LOH_N) with reliable sequencing quality and homozygous population frequency, that is, screening for variants that neither parent has.

[0079] The second screening marker module 200 performs a screening marker (abbreviated as LOH_Pat) for a variation that is only inherited from the father and has reliable sequencing quality and homozygous population frequency.

[0080] The third screening marker module 300 performs screening of variant markers (abbreviated as LOH_Mat) that are only inherited from the mother and have reliable sequencing quality and homozygous population frequency.

[0081] The fourth screening marker module 400 performs screening of variant markers (abbreviated as LOH_Pat_Mat) that are common to both parents and have reliable sequencing quality and homozygous population frequency.

[0082] Furthermore, the reliable sequencing quality means that the ratio of the total number of bases obtained by sequencing to the genome size is not less than 25X.

[0083] Furthermore, when homozygous variants inherited only from the father or homozygous variants inherited only from the mother appear consecutively with the variant markers, it indicates heterozygous deletion of a long segment or the entire chromosome or a uniparental inheritance segment or uniparental diploidy, which in turn indicates a decrease in heterozygosity.

[0084] Specifically, the first marker screening module performs screening of markers for emerging variants with reliable sequencing quality and homozygous population frequency, specifically:

[0085] Set S1 conditions: the heterozygous or homozygous column of the proband's variant site (abbreviated as Het / Hom_proband) is equal to homozygous (abbreviated as Hom), the population frequency of the public database (abbreviated as fmax) is less than or equal to 0.05, the coverage depth of the proband's variant site (abbreviated as Alt-Cover_proband) is equal to or greater than 25, the local database frequency (abbreviated as local) is less than 0.05, the filter label of the proband's GATK (abbreviated as Filter_proband) is equal to PASS, the heterozygous or homozygous column of the father's variant site (abbreviated as Het / Hom_father) is empty, and the heterozygous or homozygous column of the mother's variant site (abbreviated as Het / Hom_mother) is empty.

[0086] Variants that meet the S1 criteria are screened, and the selected variants are marked as new variants with reliable sequencing quality and homozygous population frequency.

[0087] The second screening marker module 200 performs screening markers on the variants that are only inherited from the father and have reliable sequencing quality and homozygous population frequency, specifically:

[0088] Set S2 conditions: the heterozygous or homozygous column of the proband's variant site (abbreviated as Het / Hom_proband) is equal to homozygous (abbreviated as Hom), the population frequency of the public database (abbreviated as fmax) is less than or equal to 0.05, the coverage depth of the proband's variant site (abbreviated as Alt-Cover_proband) value is equal to or greater than 25, the local database frequency (abbreviated as local) is less than 0.05, the filter label of the proband's GATK (abbreviated as Filter_proband) is equal to PASS, the heterozygous or homozygous column of the father's variant site (abbreviated as Het / Hom_father) is not a null value, and the heterozygous or homozygous column of the mother's variant site (abbreviated as Het / Hom_mother) is a null value.

[0089] Variants that meet the S2 criteria are screened, and the selected variants are marked as variants that are only inherited from the father with reliable sequencing quality and homozygous population frequency.

[0090] The third screening marker module 300 performs screening markers on the variants that are only inherited from the mother and have reliable sequencing quality and homozygous population frequency, specifically:

[0091] Set S3 conditions: the proband's variant site heterozygous or homozygous column (abbreviated as Het / Hom_proband) is equal to homozygous (abbreviated as Hom), the public database population frequency (abbreviated as fmax) is less than or equal to 0.05, the proband's variant site coverage depth (abbreviated as Alt-Cover_proband) is equal to or greater than 25, the local database frequency (abbreviated as local) is less than 0.05, the proband's GATK filter label (abbreviated as Filter_proband) is equal to PASS, the father's variant site heterozygous or homozygous column (abbreviated as Het / Hom_father) is a null value, and the mother's variant site heterozygous or homozygous column (abbreviated as Het / Hom_mother) is not a null value.

[0092] Variants that meet the S3 criteria are screened, and the selected variants are marked as variants that are only inherited from the mother with reliable sequencing quality and homozygous population frequency.

[0093] The fourth screening marker module 400 performs screening markers for variants that are both present in both parents and have reliable sequencing quality and homozygous population frequency, specifically:

[0094] Set S4 conditions: the proband's variant site heterozygous or homozygous column (abbreviated as Het / Hom_proband) is equal to homozygous (abbreviated as Hom), the public database population frequency (abbreviated as fmax) is less than or equal to 0.05, the proband's variant site coverage depth (abbreviated as Alt-Cover_proband) is equal to or greater than 25, the local database frequency (abbreviated as local) is less than 0.05, the proband's GATK filter label (abbreviated as Filter_proband) is equal to PASS, the father's variant site heterozygous or homozygous column (abbreviated as Het / Hom_father) is not a null value, and the mother's variant site heterozygous or homozygous column (abbreviated as Het / Hom_mother) is not a null value.

[0095] Variants that meet the S4 criteria are screened, and the selected variants are marked as variants that have reliable sequencing quality and homozygous population frequency and are present in both parents.

[0096] Specifically, the screening condition of population frequency was set to be less than 0.05. If the population frequency was less than 0.05, it was considered that the variant supported evidence of benignity.

[0097] Screening marker description: The markers LOH_N and LOH_Pat_Mat that appear at intervals are of little significance. When they appear consecutively with LOH_Pat or LOH_Mat markers and mutations, they indicate heterozygous deletion of long segments or whole chromosomes or uniparental inheritance segments or uniparental diploidy, which in turn indicates reduced heterozygosity. In order to increase the true positive rate, the screening setting of the population frequency is preferably less than 0.05, and a population frequency less than this is considered to be evidence that the variant supports benignity.

[0098] Aiming at the essence of reduced heterozygosity, the present invention innovatively performs heterozygosity reduction risk screening and genetic source marking on three family samples. The number of markers is small, and rapid in situ analysis is performed in the original high-throughput data table, which facilitates high-throughput analysis and helps the rapid analysis of big data.

[0099] According to the high-throughput sequencing heterozygosity reduction risk annotation method and system of the embodiment of the present invention, the high-throughput sequencing data is directly marked for the risk of heterozygosity reduction and the corresponding sites are marked for the genetic source, so that the analysis of heterozygosity reduction is intuitive and the analysis efficiency is improved. The present invention screens the sites of reduced heterozygosity and marks the sources of variation of three samples in the family. The marking items are few and the logic is clear. The true positive results of the pathogenic causes of reduced heterozygosity can be quickly identified, and the low-risk samples can be easily predicted and missed, which provides convenience for the analysis of the pathogenic causes of reduced heterozygosity, enriches the analysis method of high-throughput annotation data, and improves the detection rate.

[0100] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0101] Although the embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and cannot be understood as limiting the present invention. Those skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A high-throughput sequencing heterozygosity reduction risk annotation method, characterized in that: The proband loci of the three samples of the family high-throughput sequencing data were screened for heterozygosity reduction risk and genetic source markers were performed, including risk markers for the homozygous loci of the proband; and the genetic sources of the loci were simultaneously screened and marked, and the simultaneous screening of the genetic sources of the loci was specifically as follows: screening markers for new variants with reliable sequencing quality and homozygous population frequency, that is, screening markers for variants that neither homozygous parent has; screening markers for variants that are only inherited from the father with reliable sequencing quality and homozygous population frequency; screening markers for variants that are only inherited from the mother with reliable sequencing quality and homozygous population frequency; screening markers for variants that both parents have with reliable sequencing quality and homozygous population frequency; The screening markers for the novel variants with reliable sequencing quality and homozygous population frequency are specifically: Set S1 conditions: the heterozygous or homozygous column of the proband's variant site is equal to homozygous, the public database population frequency is less than or equal to 0.05, the proband's variant site coverage depth is equal to or greater than 25, the local database frequency is less than 0.05, the proband's GATK filter label is equal to PASS, the father's variant site heterozygous or homozygous column is empty, and the mother's variant site heterozygous or homozygous column is empty; Variants that meet the S1 criteria are screened, and the selected variants are marked as new variants with reliable sequencing quality and homozygous population frequency; The specific screening markers for the variation that is only inherited from the father and has reliable sequencing quality and homozygous population frequency are: Set S2 conditions: the heterozygous or homozygous column of the proband's variant site is equal to homozygous, the population frequency of the public database is less than or equal to 0.05, the coverage depth of the proband's variant site is equal to or greater than 25, the frequency of the local database is less than 0.05, the proband's GATK filter label is equal to PASS, the heterozygous or homozygous column of the father's variant site is not a null value, and the heterozygous or homozygous column of the mother's variant site is a null value; Variants that meet the S2 criteria are screened, and the selected variants are marked as variants that are only inherited from the father with reliable sequencing quality and homozygous population frequency; The screening markers for the variants that are only inherited from the mother and have reliable sequencing quality and homozygous population frequency are specifically: Set S3 conditions: the heterozygous or homozygous column of the proband's variant site is equal to homozygous, the public database population frequency is less than or equal to 0.05, the proband's variant site coverage depth is equal to or greater than 25, the local database frequency is less than 0.05, the proband's GATK filter label is equal to PASS, the father's variant site heterozygous or homozygous column is empty, and the mother's variant site heterozygous or homozygous column is not empty; Variants that meet the S3 criteria are screened, and the selected variants are marked as variants that have reliable sequencing quality and homozygous population frequency and are only inherited from the mother; The screening markers for the variants that have reliable sequencing quality and homozygous population frequency and that both parents have are specifically: Set S4 conditions: the heterozygous or homozygous column of the proband's variant site is equal to homozygous, the population frequency of the public database is less than or equal to 0.05, the coverage depth of the proband's variant site is equal to or greater than 25, the frequency of the local database is less than 0.05, the proband's GATK filter label is equal to PASS, the heterozygous or homozygous column of the father's variant site is not a null value, and the heterozygous or homozygous column of the mother's variant site is not a null value; Variants that meet the S4 criteria are screened, and the selected variants are marked as variants that are present in both parents with reliable sequencing quality and homozygous population frequency; The reliable sequencing quality specifically means that the ratio of the total amount of bases obtained by sequencing to the genome size is not less than 25X; The screening condition of the population frequency is set to be less than 0.05; If a homozygous variant inherited only from the father or a homozygous variant inherited only from the mother and the variant marker appear continuously, it indicates a heterozygous deletion of a long segment or the entire chromosome or a uniparental inherited segment or uniparental diploidy, which in turn indicates a decrease in heterozygosity.

2. A high-throughput sequencing heterozygosity reduction risk annotation system, characterized in that: The system is used to perform heterozygosity reduction risk screening and genetic source marking on the proband loci of the three-sample high-throughput sequencing data of the family, including: risk marking on the homozygous loci of the proband and simultaneous screening marking of the genetic source of the loci, wherein the high-throughput sequencing heterozygosity reduction risk annotation system includes: a first screening marker module, a second screening marker module, a third screening marker module and a fourth screening marker module, and the screening and marking are: The first screening marker module performs screening markers for new variants with reliable sequencing quality and homozygous population frequency, that is, screening markers for variants that neither of the homozygous parents has; The second screening marker module performs screening of variant markers that are only inherited from the father and have reliable sequencing quality and homozygous population frequency; The third screening marker module performs screening of variant markers that are only inherited from the mother and have reliable sequencing quality and homozygous population frequency; The fourth screening marker module performs screening for variants that are common to both parents with reliable sequencing quality and homozygous population frequency; the first screening marker module performs screening for new variants with reliable sequencing quality and homozygous population frequency, specifically: Set S1 conditions: the heterozygous or homozygous column of the proband's variant site is equal to homozygous, the public database population frequency is less than or equal to 0.05, the proband's variant site coverage depth is equal to or greater than 25, the local database frequency is less than 0.05, the proband's GATK filter label is equal to PASS, the father's variant site heterozygous or homozygous column is empty, and the mother's variant site heterozygous or homozygous column is empty; Variants that meet the S1 criteria are screened, and the selected variants are marked as new variants with reliable sequencing quality and homozygous population frequency; The second screening marker module performs screening markers on the variants that are only inherited from the father and have reliable sequencing quality and homozygous population frequency, specifically: Set S2 conditions: the heterozygous or homozygous column of the proband's variant site is equal to homozygous, the population frequency of the public database is less than or equal to 0.05, the coverage depth of the proband's variant site is equal to or greater than 25, the frequency of the local database is less than 0.05, the proband's GATK filter label is equal to PASS, the heterozygous or homozygous column of the father's variant site is not a null value, and the heterozygous or homozygous column of the mother's variant site is a null value; Variants that meet the S2 criteria are screened, and the selected variants are marked as variants that are only inherited from the father with reliable sequencing quality and homozygous population frequency; The third screening marker module performs screening markers on the variants that are only inherited from the mother and have reliable sequencing quality and homozygous population frequency, specifically: Set S3 conditions: the heterozygous or homozygous column of the proband's variant site is equal to homozygous, the public database population frequency is less than or equal to 0.05, the proband's variant site coverage depth is equal to or greater than 25, the local database frequency is less than 0.05, the proband's GATK filter label is equal to PASS, the father's variant site heterozygous or homozygous column is empty, and the mother's variant site heterozygous or homozygous column is not empty; Variants that meet the S3 criteria are screened, and the selected variants are marked as variants that have reliable sequencing quality and homozygous population frequency and are only inherited from the mother; The fourth screening marker module performs screening markers for the variants that are both present in parents with reliable sequencing quality and homozygous population frequency, specifically: Set S4 conditions: the heterozygous or homozygous column of the proband's variant site is equal to homozygous, the population frequency of the public database is less than or equal to 0.05, the coverage depth of the proband's variant site is equal to or greater than 25, the frequency of the local database is less than 0.05, the proband's GATK filter label is equal to PASS, the heterozygous or homozygous column of the father's variant site is not a null value, and the heterozygous or homozygous column of the mother's variant site is not a null value; Variants that meet the S4 criteria are screened, and the selected variants are marked as variants that are present in both parents with reliable sequencing quality and homozygous population frequency; The reliable sequencing quality specifically means that the ratio of the total amount of bases obtained by sequencing to the genome size is not less than 25X; The screening condition of the population frequency is set to be less than 0.05; If a homozygous variant inherited only from the father or a homozygous variant inherited only from the mother and the variant marker appear continuously, it indicates a heterozygous deletion of a long segment or the entire chromosome or a uniparental inherited segment or uniparental diploidy, which in turn indicates a decrease in heterozygosity.

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