Methods for detecting loss of heterozygosity by low-depth genome sequencing

Abstract

The present application provides methods for detecting absence of heterozygosity (AOH), such as copy number neutral loss of heterozygosity (CN-LOH), in a biological sample from an individual, as well as computer-readable media and devices for implementing the methods.

Description

[0001] Citation of Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 894,497, filed on August 30, 2019, the contents of which are hereby incorporated by reference in their entirety for all purposes. Field of the Invention

[0003] The present application relates generally to the fields of molecular genetics and molecular biology. In particular, the present application provides methods and tools for detecting absence of heterozygosity (AOH) in an individual. Background Art

[0004] Absence of heterozygosity (AOH) is a genomic alteration that causes human disease, including congenital diseases [1, 2] and tumors [3, 4], due to the loss of wild-type or imprinted genomic sequences. In addition to heterozygous loss events, AOH often presents as copy number neutral events, which represent one or more continuous stretches of homozygosity [5] and evidence of homology (e.g., parental consanguinity) or uniparental disomy (UPD) [6]. When UPD occurs on a chromosome known to harbor an imprinted gene (chromosome 6, 7, 11, 14, 15, or 20), the incidence of human disease caused by it is estimated to be 1 / 5000 in live births [7, 8]. For example, approximately 25% of cases with Prader-Willi syndrome (OMIM #: 176270) are due to maternal UPD of chromosome 15 caused by AOH or uniparental disomy [9, 10], in which both alleles of the same chromosomal region are inherited from one parent.

[0005] In routine clinical settings, chromosomal microarray analysis (CMA) using single nucleotide polymorphism (SNP) probes is the gold standard for identifying AOH with a resolution of >5 Mb [5, 6]. Currently, due to breakthroughs in molecular technologies in recent years (such as second-generation sequencing), exome sequencing (ES) has been used for clinical diagnostic testing [11-16], and researchers have begun to study AOH by using the detection of single nucleotide variants (SNVs) [17, 18]. Compared with genome sequencing (GS), ES shows limitations in the detection of copy number variants (CNVs) and even SNVs due to capture bias [6, 19]. However, despite the advantages of GS, considering the cost that patients can afford, the current clinical methods are based on low-depth (low coverage) GS, with sequencing depth ranging from about 0.1 to >5 times. Recent studies have shown that low-depth GS can identify CNVs [20-22] and chromosomal structural rearrangements [23-25], but AOH cannot be detected from current analytical methods. Furthermore, it is unclear whether uniparental heterodisomy can be detected based on the current low-depth GS.

[0006] There is a need in the art for new methods of detecting AOH, particularly by utilizing low depth GS. SUMMARY OF THE INVENTION

[0008] In a first aspect, the present application provides a method for detecting absence of heterozygosity (AOH), such as copy number neutral loss of heterozygosity (CN-LOH), in a biological sample from an individual, the method comprising:

[0009] (i) receiving low-depth sequence reads of genomic DNA from a biological sample;

[0010] (ii) aligning the sequence reads to a human genome reference, and selecting and sorting the sequence reads aligned to the human genome reference based on the aligned chromosome and genome coordinates;

[0011] (iii) identifying single nucleotide variations (SNVs) in the aligned sequence reads, wherein the SNV at each site has a mutant base type that is different from the base type at the corresponding site in the human genome reference;

[0012] (iv) identifying a homozygous SNV, a diploid heterozygous SNV, or a non-diploid heterozygous SNV from the SNVs identified in step (iii), wherein

[0013] A homozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference,

[0014] A diploid heterozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being no less than 25% and no more than 75%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference,

[0015] defining a non-diploid heterozygous SNV based on a percentage of sequence reads supporting a mutant base type being less than 25% and greater than 0% or greater than 75% and less than 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference;

[0016] (v) determining, for the window, a ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV identified in step (iv), wherein the ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV represents the ratio of the number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs of the window to the average number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs among all windows in the biological sample; and

[0017] (vi) comparing the ratio of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs of each window determined by step (v) with the average ratio of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs of the corresponding each window established from a control population.

[0018] In a second aspect, the present application provides a computer system for detecting absence of heterozygosity (AOH) (e.g., copy number neutral loss of heterozygosity (CN-LOH)) in a biological sample from an individual, the computer system comprising a processor and a memory storing a plurality of instructions, wherein the processor is configured to:

[0019] (i) receiving a low-depth sequence read of genomic DNA from the biological sample;

[0020] (ii) aligning the sequence reads to a human genome reference, and selecting and sorting the sequence reads aligned to the human genome reference based on the aligned chromosome and genome coordinates;

[0021] (iii) identifying single nucleotide variations (SNVs) in the aligned sequence reads, wherein the SNV at each site has a mutant base type that is different from the base type at the corresponding site of the human genome reference;

[0022] (iv) identifying a homozygous SNV, a diploid heterozygous SNV, or a non-diploid heterozygous SNV from the SNVs identified in (iii), wherein

[0023] A homozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference,

[0024] A diploid heterozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being no less than 25% and no more than 75%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference,

[0025] defining a non-diploid heterozygous SNV based on a percentage of sequence reads supporting a mutant base type being less than 25% and greater than 0% or greater than 75% and less than 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference;

[0026] (v) determining, for the window, a ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV identified in (iv), wherein the ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV represents the ratio of the number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs of the window to the average number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs among all windows in the biological sample; and

[0027] (vi) comparing the ratio of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs of each window determined by (v) with the average ratio of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs of the corresponding each window established from a control population.

[0028] In a third aspect, the present application provides a computer-readable medium storing a plurality of instructions, wherein the plurality of instructions, when executed by one or more processors, implement operations including the following:

[0029] (i) receiving low-depth sequence reads of genomic DNA from a biological sample of an individual;

[0030] (ii) aligning the sequence reads to a human genome reference, and selecting and sorting the sequence reads aligned to the human genome reference based on the aligned chromosome and genome coordinates;

[0031] (iii) identifying single nucleotide variations (SNVs) in the aligned sequence reads, wherein the SNV at each site has a mutant base type that is different from the base type at the corresponding site of the human genome reference;

[0032] (iv) identifying a homozygous SNV, a diploid heterozygous SNV, or a non-diploid heterozygous SNV from the SNVs identified in (iii), wherein

[0033] A homozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference,

[0034] A diploid heterozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being no less than 25% and no more than 75%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference,

[0035] defining a non-diploid heterozygous SNV based on a percentage of sequence reads supporting a mutant base type being less than 25% and greater than 0% or greater than 75% and less than 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference;

[0036] (v) determining, for the window, a ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV identified in (iv), wherein the ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV represents the ratio of the number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs of the window to the average number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs among all windows in the biological sample; and

[0037] (vi) comparing the ratio of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs of each window determined by (v) with the average ratio of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs of the corresponding each window established from a control population.

[0038] In a fourth aspect, the present application provides an apparatus comprising one or more processors and the computer-readable medium described in the third aspect.

[0039] BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The workflow of the method for detecting absence of heterozygosity (AOH) according to an exemplary embodiment of the present application is shown.

[0041] Figure 2 .Correlation of different parameters between GS (30 times sequencing depth, hereinafter referred to as GS) and low depth GS (about 4 times sequencing depth, hereinafter referred to as low depth GS) in sample HG00514. (a) Correlation between parental genome differences (Y-axis) and the ratio of heterozygous SNVs (X-axis) in GS data. (b) Correlation between parental genome differences (Y-axis) and the ratio of homozygous SNVs (X-axis) in GS data. (c) Correlation between the ratio of homozygous SNVs (Y-axis) and the ratio of diploid heterozygous SNVs (X-axis) in GS data. (d) Correlation between the ratio of diploid heterozygous SNVs indicated by low depth GS (Y-axis) and the ratio of diploid heterozygous SNVs calculated from GS data (X-axis). (e) Correlation between the ratio of homozygous SNVs indicated by low depth GS (Y-axis) and the ratio of homozygous SNVs calculated from GS data (X-axis). (f) Correlation between the ratio of homozygous SNVs (Y-axis) and the ratio of diploid heterozygous SNVs (X-axis) in low-depth GS data. In each figure, the P value of the Pearson correlation coefficient is shown in red.

[0042] Figure 3.Accuracy of AOH detection. (a) The consistency of AOH detection between GS and low-depth GS, and (b) the sensitivity and specificity of detecting AOH by low-depth GS using the detection results from GS when the increased rate of homozygous SNVs and the decreased rate of heterozygous SNVs are incorporated. 100% sensitivity and specificity were observed at a resolution of 1.4Mb. (c) The consistency of AOH detection in five cases in two independent experiments using low-depth GS, and (d) the sensitivity and specificity of detecting AOH in data from the second batch of five samples by using the data from the first batch as a reference. In each figure, the X-axis represents the size of the detected AOH. The Y-axis in Figures (a) and (c) represents the number of AOH detected, and the Y-axis in Figures (b) and (d) reflects the sensitivity and specificity of setting different detection resolution cutoffs.

[0043] Figure 4 .Detection of AOH in chromosome 5 of sample HG00733. (a) Distribution of copy numbers (indicated by black dots) between windows in chromosome 5 in this sample. The only deletion is shown by a purple arrow. The X-axis represents the genomic position in all figures, while the Y-axis represents the copy number in figure (a). Distribution (b) of the standardized ratio of heterozygous SNVs on chromosome 5 and the standardized ratio of homozygous SNVs (c) by low-depth GS method. Distribution (e) of the ratio of heterozygous SNVs on chromosome 5 and the ratio of homozygous SNVs (f) by GS method. In figures (b) and (d), AOH is identified by observing the continuously decreasing ratio of heterozygous SNVs (indicated by red arrows, where the number of windows is included at the bottom). In figures (c) and (e), there are regions with continuously increasing homozygous SNV ratios (indicated by blue arrows, where the number of windows is included at the bottom). (f) Distribution of parental genomic differences on chromosome 5. The Y-axis in figures (bf) shows the ratio of each corresponding parameter. The genomic region of the large AOH-seq [GRCh37] 5q23q34 (149200000_164900000) x 2 hmz is shown by a pair of green dashed lines in each figure.

[0044] Figure 5.AOH was detected in sample 18C1564. Copy number distribution (a) and genotype distribution (b) reported by CMA. The X-axis in Figures (a) and (b) represents the genomic position. The Y-axis in Figure (a) represents the log2 ratio of the copy number, and the Y-axis in Figure (b) represents the distribution of different numbers of genotypes: 0, 1, 2, and 3 represent the genotype as A allele, AB, B, and AAB / ABB, respectively. In Figure (a), each dot represents a probe, and the copy ratios classified as gain, neutrality, or loss are shown in blue, black, and red, respectively. In Figure (b), the presence of each genotype is shown as a green dot in the corresponding line, and the reported regions with AOH are highlighted with a green background. Two additional AOH reported by low-depth GS (not reported by CMA) are highlighted with a yellow background, and the absence of heterozygous genotype (AB) in these two regions is indicated by two red arrows. (c) Copy number distribution reported by low-depth GS, with windows indicated by black dots. The X-axis in Figure (cg) represents the genomic position on chromosome 6, and in Figure (c), the Y-axis represents the copy number. Figures (d) to (f) respectively show the ratio distribution of "germline" heterozygous SNV (AB), homozygous SNV and "mosaic" heterozygous SNV (AAB / ABB). In Figure (d), the candidate region where AOH is detected is indicated by each pair of red arrows and the number of windows, and in Figure (e), the windows with the homozygous SNV ratio increased in those regions reported in Figure (d) are shown by each pair of blue arrows and the number of windows. The two cryptic regions reported only by low depth GS highlighted in Figure (b) are also highlighted in Figure (de). In Figure (f), the candidate region with increased "mosaic" heterozygous SNV ratio is shown by each pair of blue arrows and the number of windows. In Figure (g), the Y-axis shows the maternal inherited genotype (black dots) in the upper line and the paternal inherited genotype (black dots) in the lower line. If the ratio of maternal / paternal genotypes is greater than 5, the middle line is displayed in red, and if the ratio is less than 0.2, the middle line is displayed in blue.

[0045] Figure 6.AOH was detected in a mosaic trisomy event in sample 18C1493. Copy number distribution (a) and genotype distribution (b) reported by CMA. In Figures (a) and (b), the X-axis represents the genomic position. The Y-axis in Figure (a) represents the log2 ratio of the copy number, while the Y-axis in Figure (b) represents the distribution of different numbers of genotypes: 0, 1, 2, and 3 represent the genotype as A allele, AB, B, and AAB / ABB, respectively. In Figure (a), each dot represents a probe, and the copy ratios classified as increased, neutral, or lost are shown in blue, black, and red, respectively. An increase of approximately 40% of the entire chromosome 6 is shown (indicated by the blue box). In Figure (b), the presence of each genotype is shown as a green dot in the corresponding line, and the reported region with AOH is highlighted with a green background. (c) Copy number distribution reported by low-depth GS, with the window indicated by a black dot. The results confirmed that the entire chromosome 6 increased by approximately 40% (indicated by the blue line). The X-axis in Figure (cg) represents the genomic position on chromosome 6, and in Figure (c), the Y-axis represents the copy number. Figures (d) to (f) respectively show the ratio distribution of " germline " heterozygous SNV (AB), homozygous SNV and " mosaic " heterozygous SNV (AAB / ABB). In Figure (d), the candidate region for detecting AOH is indicated by every pair of red arrows and window quantity, and in Figure (e), the window with the homozygous SNV ratio of improvement in those regions reported in Figure (d) is shown by every pair of blue arrows and window quantity. In Figure (f), the candidate region with the " mosaic " heterozygous SNV ratio of increase is shown by every pair of blue arrows and window quantity. In Figure (g), the Y-axis shows the maternal genetic genotype (black dots) in the upper line and the paternal genetic genotype (black dots) in the lower line. If the ratio of maternal / paternal genotype is greater than 5, the middle line is shown in red, and if the ratio is less than 0.2, the middle line is shown in blue.

[0046] Figure 7 .Cryptic AOH reported by low-depth GS. Figures (a), (c) and (e) show the copy number distribution in 17C1122, 17C1175 and 17C1176, respectively, while figures (b), (d) and (f) show the ratio distribution of heterozygous SNVs in each of these three samples. The X-axis in each figure represents the genomic position. The Y-axis in figures (a), (c) and (e) represents the copy number, while the Y-axis in figures (b), (d) and (f) represents the ratio of heterozygous SNVs. In figures (b), (d) and (f), the candidate regions with AOH reported by low-depth GS are indicated by each pair of red arrows and the number of windows involved at the bottom. The green dashed line shows the region of the KCTD7 gene on each figure, while in figure (b), the cryptic AOH is highlighted with a yellow background.

[0047] Figure 8.Correlation of different parameters between GS and low-depth GS in HG00733 samples. (a) Correlation between parental genome differences (Y-axis) and the ratio of heterozygous SNVs (X-axis) in GS data. (b) Correlation between parental genome differences (Y-axis) and the ratio of homozygous SNVs (X-axis) in GS data. (c) Correlation between the ratio of homozygous SNVs (Y-axis) and the ratio of heterozygous SNVs (X-axis) in GS data. (d) Correlation between the ratio of heterozygous SNVs indicated by low-depth GS (Y-axis) and the ratio of heterozygous SNVs calculated by GS data (X-axis). (e) Correlation between the ratio of homozygous SNVs indicated by low-depth GS (Y-axis) and the ratio of homozygous SNVs calculated by GS data (X-axis). (f) Correlation between the ratio of homozygous SNVs (Y-axis) and the ratio of heterozygous SNVs (X-axis) in low-depth GS data. In each figure, the P value of the Pearson correlation coefficient is shown in red.

[0048] Fig. 9 .Observations of reduced heterozygous SNV ratios in regions with heterozygous deletions. A heterozygous deletion arr[GRCh37]1q23.1q25.2(158043081_176445395)x1 dn was reported in sample 18C0241. (a) Distribution of copy number between windows in chromosome 5 in this sample (indicated by black dots). In all figures, the X-axis represents genomic position and the Y-axis represents copy number in Figure (a). In Figure (a), large deletions are shown with a pair of lines and arrows for the affected bands. In the same sample, the distribution of standardized ratios of heterozygous SNVs in low-depth GS (b) and the distribution of ratios of heterozygous SNVs (c) both show reduced ratios in copy number deletion regions. In Figures (b) and (c), regions with reduced continuous heterozygous SNV ratios are indicated by red arrows, and the bottom includes the number of windows.

[0049] Fig.10.Detection of AOH in chromosome 2 of HG00733 sample. (a) Distribution of copy numbers between windows (represented by black dots) in chromosome 2 in this sample. The only deletion is shown by a purple arrow. In all figures, the X-axis represents the genomic position, and the Y-axis represents the copy number in Figure (a). By low-depth GS method, the distribution of the standardized ratio of heterozygous SNVs on chromosome 2 (b) and the distribution of the standardized ratio of homozygous SNVs (c). By GS, the distribution of the ratio of heterozygous SNVs on chromosome 2 (e) and the distribution of the ratio of homozygous SNVs (f). In Figures (b) and (d), AOH is identified by observing the decreasing ratio of continuous heterozygous SNVs (represented by red arrows, where the number of windows is included at the bottom). In Figures (c) and (e), there are regions with increasing ratios of continuous homozygous SNVs (represented by blue arrows, where the number of windows is included at the bottom). (f) Distribution of parental genomic differences on chromosome 2. The Y-axis in Figures (bf) shows the ratio of each corresponding parameter. The genomic region of the large AOH seq [GRCh37] 2p23.2p21 (29700000_42600000) x 2 hmz is shown by a pair of green dashed lines in each figure.

[0050] Fig.11 .AOH was detected in a mosaic trisomy event in sample 16C0836. (a) Copy number distribution reported by low-depth GS, with windows indicated by black dots. The results confirmed that the entire chromosome 7 was increased by approximately 40% (indicated by the blue line). The X-axis in Figures (ad) represents the genomic position on chromosome 7, and in Figure (a), the Y-axis represents the copy number. Figures (b) to (d) show the ratio distribution of "germline" heterozygous SNVs (AB), homozygous SNVs, and "mosaic" heterozygous SNVs (AAB / ABB), respectively. In Figure (b), candidate regions where AOH was detected are indicated by each pair of red arrows and the number of windows, while in Figure (c), windows with increased ratios of homozygous SNVs in those regions reported in Figure (b) are shown by each pair of blue arrows and the number of windows. In Figure (d), candidate regions with increased ratios of "mosaic" heterozygous SNVs are shown by each pair of blue arrows and the number of windows.

[0051] Fig.12.AOH was detected in sample aCGH15274. (a) Copy number distribution reported by low-depth GS, with windows indicated by black dots. The x-axis in panels (ae) indicates genomic position on chromosome 6, and in panel (a), the Y-axis indicates copy number. Panels (b) to (d) show the distribution of the ratios of "germline" heterozygous SNVs (AB), homozygous SNVs, and "mosaic" heterozygous SNVs (AAB / ABB), respectively. In panel (b), candidate regions where AOH was detected are indicated by each pair of red arrows and the number of windows, and in panel (c), windows with increased ratios of homozygous SNVs in those regions reported in panel (b) are shown by each pair of blue arrows and the number of windows. In panel (d), candidate regions with increased ratios of "mosaic" heterozygous SNVs are shown by each pair of blue arrows and the number of windows. In panel (e), the Y-axis shows the maternally inherited genotypes (black dots) in the upper line and the paternally inherited genotypes (black dots) in the lower line. If the ratio of maternal / paternal genotypes is greater than 5, the middle line is displayed in red, and if the ratio is less than 0.2, the middle line is displayed in blue.

[0052] Fig.13 .Distribution of the ratios of different types of SNVs in deletions and duplications. (a) Copy number distribution reported by low-depth GS, with windows indicated by black dots. CNV analysis results showed deletion seq [GRCh37]del(8)(p23.3p23.2)chr8:G.101345523520del and duplication seq [GRCh37]dup(8)(q22.1q24.3)chr8:G.98620704_146298884dup in 17BA0551. The X-axis in (ad) represents the genomic position on chromosome 8, while in (a), the Y-axis represents the copy number. (b) to (d) show the ratio distributions of "germline" heterozygous SNVs (AB), homozygous SNVs, and "mosaic" heterozygous SNVs (AAB / ABB), respectively. In (b), candidate regions with decreased ratios of "germline" heterozygous SNVs are indicated by each pair of red arrows and window numbers, while in (c), windows with increased ratios of homozygous SNVs in those regions reported in (b) are indicated by each pair of blue arrows and window numbers. In (d), candidate regions with increased ratios of "mosaic" heterozygous SNVs are indicated by each pair of blue arrows and window numbers. The results show that all ratios decreased in the case of 8p terminal deletions, while the ratios of "mosaic" heterozygous SNVs increased in the case of 8q terminal duplications.

[0053] DETAILED DESCRIPTION OF THE INVENTION

[0054] Existing AOH detection methods usually require sequencing from targeted sequencing (e.g., exome sequencing) or genome sequencing (GS) (e.g., >30 times sequencing depth). Targeted sequencing methods can only be applied to specific regions of the genome, while GS methods are expensive for clinical practice.

[0055] There is no report on AOH detection using low-depth methods. Ideally, the principle of AOH detection is to identify regions with a common base type or expressed as a homozygous base type. Those skilled in the art generally understand that for low-depth methods, it may be difficult to determine whether a site is truly a bi-allelic mutation (homozygous) or a reference allele deletion caused by sequencing bias. At the same time, "heterozygous SNVs" are detected in regions with AOH, which is attributed to a high probability of misalignment. However, when there is a region with AOH, the ratio of "heterozygous SNVs" will decrease. The inventors of the present application have developed a method for detecting AOH using low-depth GS, which uses the ratio of heterozygous SNVs on the genome or chromosome instead of identifying the absence of heterozygous base types or AB alleles, thereby completing the various inventions described in the present application.

[0056] In a first aspect, the present application provides a method for detecting absence of heterozygosity (AOH), such as copy number neutral loss of heterozygosity (CN-LOH), in a biological sample from an individual, the method comprising:

[0057] (i) receiving low-depth sequence reads of genomic DNA from a biological sample;

[0058] (ii) aligning the sequence reads to a human genome reference, and selecting and sorting the sequence reads aligned to the human genome reference based on the aligned chromosome and genome coordinates;

[0059] (iii) identifying single nucleotide variations (SNVs) in the aligned sequence reads, wherein the SNV at each site has a mutant base type that is different from the base type at the corresponding site in the human genome reference;

[0060] (iv) identifying a homozygous SNV, a diploid heterozygous SNV, or a non-diploid heterozygous SNV from the SNVs identified in step (iii), wherein

[0061] A homozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference,

[0062] A diploid heterozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being no less than 25% and no more than 75%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference,

[0063] defining a non-diploid heterozygous SNV based on a percentage of sequence reads supporting a mutant base type being less than 25% and greater than 0% or greater than 75% and less than 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference;

[0064] (v) determining, for the window, a ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV identified in step (iv), wherein the ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV represents the ratio of the number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs of the window to the average number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs among all windows in the biological sample; and

[0065] (vi) comparing the ratio of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs of each window determined by step (v) with the average ratio of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs of the corresponding each window established from a control population.

[0066] In some embodiments, the biological sample is selected from peripheral blood, chorionic villi, amniotic fluid, umbilical cord blood, placental tissue, and tissue samples from organs. In some embodiments, the individual is a pregnant woman, an infant, an individual with cancer, or an individual suspected of having cancer. As will be appreciated by those skilled in the art, the detection of AOH can be used in various scenarios, such as prenatal genetic diagnosis, postnatal genetic diagnosis, or even cancer genetics. Therefore, those skilled in the art can determine candidate individuals or suitable biological samples according to the purpose of the AOH detection.

[0067] Single-end sequence reads or double-end sequence reads (also referred to as "read pairs") are well known to those skilled in the art and can be suitably used in the present application.

[0068] Compared with GS that requires sequencing, the low-depth genome sequencing in the present application can have a lower sequencing depth, such as 3-5 times the sequencing depth, such as 3 times the sequencing depth.

[0069] A suitable human genome reference for the alignment step can be selected by a person skilled in the art. In a specific embodiment, the human genome reference is hg19 / GRCh37 or hg38 / GRCh38.

[0070] A suitable human genome reference for the alignment step can also be selected by those skilled in the art, including but not limited to Short Oligonucleotide Alignment Program 2 (SOAP2) or Burrows-Wheeler Alignment Program (BWA) and Bowtie 2. The default settings can be used.

[0071] In some embodiments, step (ii) further comprises removing sequence reads resulting from polymerase chain reaction (PCR) duplications.

[0072] In some embodiments, step (iii) further comprises deleting the following sites:

[0073] (a) The minimum read depth of a locus is determined by the minimum read depth of the biological sample;

[0074] (b) the maximum read depth of the locus is determined by the maximum read depth of the biological sample; or

[0075] (c) Sites where no sequence reads supported the mutant base type.

[0076] In some embodiments, the window in step (v) has a fixed length, eg, 100 kb.

[0077] In some embodiments, step (v) comprises:

[0078] determining the number of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs of the window,

[0079] determining the average number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs in all windows in the biological sample, and

[0080] The ratio of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs for the window is calculated by dividing the number of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs identified for the window by the average number of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs for all windows in the biological sample.

[0081] In some embodiments, the control population has the same sex as the individual. In some embodiments, the control population has at least 30 control individuals.

[0082] In theory, AOH is defined as loss of heterozygosity or presence of long fragment homozygosity in diploid chromosomes when the copy number is neutral (no deletion occurs). For male individuals, only the autosomes are disomic, while for female individuals, both the autosomes and sex chromosomes are disomic. Therefore, the control population can include control individuals of the same sex as the test individual.

[0083] In some embodiments, step (vi) comprises:

[0084] The ratio of the homozygous SNV, diploid heterozygous SNV or non-diploid heterozygous SNV of the window is normalized relative to the average ratio of the homozygous SNV, diploid heterozygous SNV or non-diploid heterozygous SNV of the corresponding window established from the control population, thereby providing the corresponding ratio ratio of the homozygous SNV, diploid heterozygous SNV or non-diploid heterozygous SNV of the window.

[0085] In some embodiments, in step (vi), an increased rate of non-diploid heterozygous SNVs is indicative of mosaic AOH, and preferably, step (vi) further comprises:

[0086] In the case of copy number mosaic duplication represented as a copy ratio greater than 1 or copy number neutrality represented as a copy ratio equal to 1, for all windows with a non-diploid heterozygous SNV ratio ratio greater than 1, a region is defined if there are multiple consecutive windows with a non-diploid heterozygous SNV ratio ratio greater than 1.15; and

[0087] Such regions are reported as having chimeric AOH.

[0088] In some embodiments, in step (vi), a decreased rate of diploid heterozygous SNVs and an increased rate of homozygous SNVs is indicative of AOH, and preferably, step (vi) further comprises:

[0089] In the case where copy number neutrality is represented as a copy ratio equal to 1, for all windows with a diploid heterozygous SNV ratio ratio less than 1, if there are multiple consecutive windows with a diploid heterozygous SNV ratio ratio less than 0.5 and the percentage of windows with a homozygous ratio ratio greater than 1.25 is at least 25%, then a region is defined, and optionally, if there is no more than one window with a diploid heterozygous SNV ratio ratio greater than 0.5 but less than 1, two regions are combined into one region; and

[0090] The area is reported as having AOH present.

[0091] In some embodiments, the average ratio of heterozygous SNVs for each window corresponding to the control population is determined by:

[0092] (ci) receiving low-depth sequence reads of genomic DNA from a biological sample of a control individual from a control population;

[0093] (cii) aligning the sequence reads with a human genome reference, and selecting and sorting the sequence reads aligned with the human genome reference based on the aligned chromosome and genome coordinates;

[0094] (ciii) identifying single nucleotide variations (SNVs) in the aligned sequence reads, wherein the SNV at each site has a mutant base type that is different from the base type at the corresponding site in the human genome reference;

[0095] (civ) identifying a homozygous SNV, a diploid heterozygous SNV, or a non-diploid heterozygous SNV from the SNVs identified in step (ciii), wherein

[0096] A homozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference,

[0097] A diploid heterozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being no less than 25% and no more than 75%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference,

[0098] defining a non-diploid heterozygous SNV based on a percentage of sequence reads supporting a mutant base type being less than 25% and greater than 0% or greater than 75% and less than 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference;

[0099] (cv) determining, for the window, a ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV identified in step (civ), wherein the ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV represents the ratio of the number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs of the window to the average number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs among all windows in the biological sample; and

[0100] (cvi) averaging the ratios of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs for the windows from all control individuals to provide an average ratio of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs for the corresponding windows of the control population.

[0101] In some embodiments, the method further comprises a sex determination step performed between steps (cii) and (ciii), wherein the alignment ratios of the X chromosome, the Y chromosome and the whole genome are calculated as the number of sequence reads aligned to the chromosome / genome respectively divided by the length defined by the human reference genome, the Y chromosome percentage is calculated as the alignment ratio of the Y chromosome divided by the alignment ratio of the whole genome, and if the Y chromosome percentage is greater than 0.05, the control individual is considered to be male.

[0102] In some embodiments, steps (ciii) to (cvi) are performed separately on male and female control individuals based on the results of the gender determination step.

[0103] In some embodiments, in step (cvi), if the ratio of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs in a window has a significant deviation between control individuals, the average ratio of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs in the window is calculated as the average of the ratios of the window and its flanking windows (e.g., two upstream windows and two downstream windows).

[0104] As a non-limiting example, the process from establishing a control dataset to detecting AOH in case samples is described below.

[0105] Create a control dataset

[0106] (i) Comparison

[0107] For each sample, single-end or double-end reads were aligned to a human genome reference (e.g., GRCh37 / hg19 or GRCh38 / hg38) using the default alignment software [i.e., Short Oligonucleotide Alignment Program 2 (SOAP2), Burrows-Wheeler Alignment Program (BWA), and Bowtie2]. All reads / read pairs aligned to the human genome reference were selected and sorted based on the aligned chromosome and coordinates, and then reads / read pairs due to polymerase chain reaction (PCR) duplications were removed. The remaining reads / read pairs were named processed reads / read pairs for further analysis.

[0108] (ii) Gender determination

[0109] The comparison ratios of X chromosome, Y chromosome and whole genome are calculated as the number of read length / read length pairs compared with specific chromosome / genome divided by length (defined by human reference genome). The Y chromosome percentage is calculated as the comparison ratio of Y chromosome divided by the comparison ratio of whole genome, and if the Y chromosome percentage is greater than 0.05, the case is considered to be male. After gender determination, a minimum of 30 cases are selected from each gender for control construction independently.

[0110] (iii) Putative single nucleotide variant (SNV) detection

[0111] The processed reads / read pairs from step (i) are used as input to identify the alignment results in each coordinate by the MPileup module from Samtools. From each site, the alignment information can be presented as:

[0112] a. "." is the base type that is consistent with the human genome reference, and the aligned strand is the positive strand or "+";

[0113] b. "," is the base type consistent with the human genome reference, and the aligned strand is the negative strand or "-";

[0114] c. "A" (using base type "A" as an example) is a mutant base type having a base type different from that of the human genome reference, and the aligned strand is the positive strand or "+";

[0115] d. "a" (using base type "A" as an example) has a mutant base type that is different from the base type of the human genome reference, and the alignment strand is the minus strand or "-".

[0116] Putative SNV calls are performed for each locus, with reference chromosome, coordinates, basetype, and alignment information, with the loci being removed as described below:

[0117] a. The minimum read depth of each "putative" site is determined by the minimum read depth of the specific sample. For example, when there is only 3 times the sequencing depth for a case, those sites with a read depth of <3 can be deleted. In addition, assuming that the sequencing read depth follows a normal distribution, those sites with extremely high read depths (e.g. > mean + 3SD (standard deviation)) can also be deleted, because they are likely caused by systematic errors; or

[0118] b. No read length supporting the mutant base type;

[0119] (iv) Homozygous or “germline” / “mosaic” heterozygous SNV ratio

[0120] A genome-wide fixed window (e.g., 100 kb) can be used. i , the number of homozygous or "germline" / "mosaic" heterozygous SNV Hi / Gi / Mi identified in step (iii) can be counted, and the average of the corresponding type of SNV in all windows in a certain sample will be counted as RH / RG / RM. i, the ratio of homozygous or "germline" / "mosaic" heterozygous SNVRHi / RGi / RMi can be calculated as Hi / Gi / Mi divided by RH / RG / RM.

[0121] To build a control dataset, for each gender, all control samples in window W i The average ratio can be calculated as the average of RHi / RGi / RMi, named NRHi / NRGi / NRMi. The average ratio of each window in all the whole genome can be retained for future population-based normalization of case samples.

[0122] Detection of AOH in case samples

[0123] (i) Data preparation

[0124] For case C, reads / read pairs were aligned, sorted, PCR duplicates removed, sex determined, putative SNV calls, and ratios of homozygous or “germline” / “mosaic” heterozygous SNVs determined.

[0125] Then, for each window W i , the ratio of homozygous or "germline" / "mosaic" heterozygous SNV NRHi / NRGi / NRMi is normalized to the mean of NRHia / NRGia / NRMia for that window from the corresponding sex control cohort (named NRHic / NRGic / NRMic). If NRHic / NRGic / NRMic has high deviation, then W i The average of NRHic / NRGic / NRMic itself together with the four flanking windows (two upstream windows and two downstream windows) is designated as W i The standardized ratio in .

[0126] (ii) Screening of candidate regions with AOH

[0127] The estimated AOH may be defined as the region / window where NRGic is less than 0.5, and the window where NRGic is less than 0.5 is selected.

[0128] (iii) Breakpoint determination

[0129] For all windows with NRGic less than 0.5, if there are multiple windows with NRGic less than 0.5 in a row, a region is defined, and the percentage of windows with NRGic greater than 1.25 should be greater than 25%. In addition, if only less than one window has NRGic greater than 0.5 but less than 1, two regions can be combined together. The final region with AOH can be reported after window / region combination. The resolution of this detection can be as small as 2.5Mb.

[0130] In a second aspect, the present application provides a computer system for detecting absence of heterozygosity (AOH) (e.g., copy number neutral loss of heterozygosity (CN-LOH)) in a biological sample from an individual, the computer system comprising a processor and a memory storing a plurality of instructions, wherein the processor is configured to:

[0131] (i) receiving a low-depth sequence read of genomic DNA from the biological sample;

[0132] (ii) aligning the sequence reads to a human genome reference, and selecting and sorting the sequence reads aligned to the human genome reference based on the aligned chromosome and genome coordinates;

[0133] (iii) identifying single nucleotide variations (SNVs) in the aligned sequence reads, wherein the SNV at each site has a mutant base type that is different from the base type at the corresponding site of the human genome reference;

[0134] (iv) identifying a homozygous SNV, a diploid heterozygous SNV, or a non-diploid heterozygous SNV from the SNVs identified in (iii), wherein

[0135] A homozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference,

[0136] A diploid heterozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being no less than 25% and no more than 75%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference,

[0137] defining a non-diploid heterozygous SNV based on a percentage of sequence reads supporting a mutant base type being less than 25% and greater than 0% or greater than 75% and less than 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference;

[0138] (v) determining, for the window, a ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV identified in (iv), wherein the ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV represents the ratio of the number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs of the window to the average number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs among all windows in the biological sample; and

[0139] (vi) comparing the ratio of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs of each window determined by (v) with the average ratio of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs of the corresponding each window established from a control population.

[0140] In a third aspect, the present application provides a computer-readable medium storing a plurality of instructions, wherein the plurality of instructions, when executed by one or more processors, implement operations including the following:

[0141] (i) receiving low-depth sequence reads of genomic DNA from a biological sample of an individual;

[0142] (ii) aligning the sequence reads to a human genome reference, and selecting and sorting the sequence reads aligned to the human genome reference based on the aligned chromosome and genome coordinates;

[0143] (iii) identifying single nucleotide variations (SNVs) in the aligned sequence reads, wherein the SNV at each site has a mutant base type that is different from the base type at the corresponding site of the human genome reference;

[0144] (iv) identifying a homozygous SNV, a diploid heterozygous SNV, or a non-diploid heterozygous SNV from the SNVs identified in (iii), wherein

[0145] A homozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference,

[0146] A diploid heterozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being no less than 25% and no more than 75%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference,

[0147] defining a non-diploid heterozygous SNV based on a percentage of sequence reads supporting a mutant base type being less than 25% and greater than 0% or greater than 75% and less than 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference;

[0148] (v) determining, for the window, a ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV identified in (iv), wherein the ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV represents the ratio of the number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs of the window to the average number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs among all windows in the biological sample; and

[0149] (vi) comparing the ratio of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs of each window determined by (v) with the average ratio of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs of the corresponding each window established from a control population.

[0150] In a fourth aspect, the present application provides an apparatus comprising one or more processors and the computer-readable medium described in the third aspect.

[0151] Features or embodiments described in the first aspect may be applied to or combined in the second to fourth aspects.

[0152] It should be understood that any embodiment of the present invention can be implemented in the form of control logic using hardware (e.g., application specific integrated circuits or field programmable gate arrays) and / or computer software using a general programmable processor with a modular or integrated manner. In this article, the processor includes a single-core processor, a multi-core processor on the same integrated chip, or a plurality of processing units on a single circuit board or networked. Based on the disclosure and teaching provided herein, a person of ordinary skill in the art can know and understand other ways and / or methods of implementing the embodiments of the present invention using hardware and a combination of hardware and software. Any software component or function described in this application can be implemented as a software code executed by a processor using any suitable computer language (e.g., Java, C, C++, C#, Objective-C, Swift or a scripting language (e.g., Perl or Python) using, for example, conventional or individual-oriented technology). The software code can be stored on a computer-readable medium as a series of instructions or commands for storage and / or transmission. Suitable non-temporary computer-readable media may include random access memory (RAM), read-only memory (ROM), magnetic media such as hard disk drives or floppy disks, or optical media such as compact disks (CDs) or DVDs (digital versatile disks), flash memory, etc. The computer readable medium may be any combination of such storage or transmission devices.

[0153] This program can also be encoded and transmitted using a carrier signal suitable for transmitting by wired, optical and / or wireless networks that meet various protocols (including the Internet). Therefore, the computer-readable medium according to an embodiment of the present invention can be created using a data signal encoded with this program. The computer-readable medium encoded with program code can be packaged together with compatible devices or provided separately from other devices (for example, via Internet download). Any such computer-readable medium can be resident on a single computer product or therein (for example, hard disk drive, CD or entire computer system), and can be present on or therein different computer products in a system or network. A computer system can include a monitor, a printer or other suitable displays for providing any result described herein to the user.

[0154] Any method described herein can be performed in whole or in part with a computer system comprising one or more processors, which can be configured to perform the steps. Therefore, embodiments can be directed to a computer system configured to perform the steps of any method described herein, possibly together with different components that perform separate steps or separate step groups. Although represented by numbered steps, the steps of the method herein can be performed simultaneously or in different orders. In addition, the parts of these steps can be used together with the parts of other steps from other methods. In addition, all or part of the steps can be optional. In addition, any step of any method can be performed with a module, unit, circuit or other device for performing these steps.

[0155] Without departing from the spirit and scope of the embodiments of the invention, the specific details of the specific embodiments may be combined in any suitable manner. However, other embodiments of the invention may be directed to specific embodiments relating to each individual aspect or specific combinations of these individual aspects.

[0156] The above description of exemplary embodiments of the present invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form described, and many modifications and variations are possible in light of the above teachings.

[0157] In the foregoing description, for the purpose of explanation, various details have been set forth in order to provide an understanding of various embodiments of the present invention technology. However, it will be readily understood by those skilled in the art that certain embodiments may be practiced without some of these details or with additional details present.

[0158] Several embodiments have been described, and those skilled in the art will recognize that various modifications, alternative structures and equivalents may be used without departing from the spirit of the present invention. In addition, in order to avoid unnecessarily obscuring the present invention, many well-known processes and elements are not described. In addition, the details of any particular embodiment may not always be present in a variation of the embodiment, or may be added to other embodiments.

[0159] Where a range of values ​​is provided, it is to be understood that, unless the context clearly specifies otherwise, each intermediate value (to one tenth of the unit of the lower limit) between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any of the values ​​or intermediate values ​​in the range and any other values ​​or intermediate values ​​in the range is encompassed. The upper and lower limits of these smaller ranges may be independently included or excluded within the range, and each range that includes either one or both limits or both limits in the smaller range is also included within the present invention, subject to any specifically excluded limits within the range. When the range includes one or both of the limits, the range excluding one or both of the included limits is also included.

[0160] Unless specifically stated to the contrary, the use of “a,” “an,” or “the” is intended to mean “one or more.” Unless specifically stated to the contrary, the use of “or” is intended to mean an inclusive or rather than an exclusive or.

[0161] All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference in their entirety for all purposes. No admission is made that any of them is prior art. Example

[0162] method

[0163] Individual registration and sample collection

[0164] GS data from a trio (proband-father-mother) from the 1000 Genomes Project

[26] [dual-end 126-bp, from the Illumina platform (San Diego, CA, USA), >30 times sequencing depth, hereinafter referred to as GS] and 50 cases with increased nuchal translucency sequenced in this study [dual-end 100-bp, from MGISEQ-2000 (MGI, BGI-Shenzhen, Shenzhen, China)] were used to develop and validate the method. In addition, 12 DNA samples from 10 AOH reported by CMA were recruited for low-depth GS (~4 times sequencing depth). Written informed consent was obtained from each participant (Table 1). Parental DNA samples were also obtained for two cases (Table 1).

[0165]

[0166]

[0167]

[0168] DNA preparation and conventional CMA

[0169] When tested, genomic DNA was extracted from chorionic villi, amniotic fluid, or fetal cord blood using the DNeasy Blood & Tissue Kit (Cat. No. 69506, Qiagen, Hilden, Germany). DNA was quantified using the Qubit dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA), and DNA integrity was assessed by agarose gel electrophoresis.

[0170] For routine CMA testing, we used a validated custom CMA 8×60k fetal DNA chip v2.0 (Agilent Technologies, Santa Clara, CA, USA) containing SNP and comparative genomic hybridization (CGH) probes [28, 29]. Experiments were performed according to the manufacturer's protocol. CNV and AOH analysis was performed using CytoGenomics software [28, 29].

[0171] Low Depth GS

[0172] 100 ng of genomic DNA from each sample was fragmented to a fragment size range of 300 to 500 bp using a Covaris S2 focused ultrasonicator (Covaris, Inc., Woburn, MA, USA). The library construction protocol included end repair, formation of fragments with A at the tail, adapter ligation, and PCR amplification. The PCR product was then heat denatured to form single-stranded DNA and then circularized with DNA ligase. After the DNA nanoballs were constructed, each sample was sequenced at 100 bp at each end on the MGISEQ-2000 platform (MGI) at a read depth of approximately 4 times the sequencing depth

[30] . To evaluate reproducibility, low-depth GS including library construction and sequencing was repeated for 5 samples (Table 1).

[0173] Data analysis and detection of SNVs

[0174] Paired-end reads were QC assessed by FastQC (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ) and subsequently aligned to the human reference genome (GRCh37 / hg19) using the Burrows-Wheeler Alignment Program (BWA)

[31] . The alignment files were converted to a different format and reads suspected to be caused by PCR duplication were removed using SAMtools

[32] . For GS, SNV detection was performed using HaplotypeCallerv3.4 from the Genome Analysis Toolkit (GATK, Broad Institute)

[33] , and homozygous and heterozygous SNVs were classified using ANNOVAR

[34] . Of note, since the SNVs detected by the GATK HaplotypeCaller module are based on a diploid setting, all heterozygous SNVs reported by GS were classified as “germline” heterozygous SNVs for further analysis.

[0175] For each set of GS (>30 times sequencing depth), low-depth GS (4 times sequencing depth) was simulated by randomly selecting paired-end reads

[24] . For low-depth GS data by computer simulation or sequencing, paired-end reads were aligned, formatted, and PCR duplicates removed as described above. The coverage of mapped reads with genotype information at each genomic site was then summarized by the mpileup module from SAMtools

[32] , and sites with reads supporting the mutant base type were selected and defined as SNVs. SNVs were classified into three categories based on the SNV allele fraction (VAF), which was calculated as the number of reads supporting the mutant base type divided by the total number of reads supporting that specific locus: (1) If no reads supported the wild-type allele (the percentage of sequence reads supporting the mutant base type was 100%), a homozygous SNV was defined; (2) If the VAF was not less than 25% and not greater than 75%, it was classified as a "germline" heterozygous SNV; (3) If the VAF was less than 25% and greater than 0% or greater than 75% and less than 100%, a "mosaic" heterozygous SNV was detected.

[0176] Calculation of parental genomic differences

[0177] For trios that downloaded GS data from the 1000 Genomes Project, genotype information from each parental sample was also obtained from GATK. The number of SNVs in which the two parents were homozygous for different genotypes was counted as P with a fixed window (100-kb size). d , and the total number of detected SNVs is also counted as P in the same window tThe ratio of parental genomic differences in each window was calculated as P d Divide by P t , and get P dr .

[0178] Ratios of different types of SNVs

[0179] For each SNV detected by GS or low-depth GS, the population-based normalized ratio of homozygous SNVs with a fixed window size of 100 kb was calculated as follows: (1) For a specific window W i , based on the number of homozygous SNVs counted at the genomic locus H i ; (2) H is then normalized relative to the average number of homozygous SNVs in all windows for that case i , set to RH i ; and (3) further normalized by the average ratio of homozygous SNVs across all cases in that specific window and set to NRH i . i The same method is used to calculate the “germline” heterozygous SNV (NRG i ) and population-based normalized ratios of “mosaic” heterozygous SNVs (NRMi).

[0180] CNV and AOH testing

[0181] CNV detection was performed based on our previous studies [22, 35]. Since an internal reference cohort was developed using data generated from a single-end read of 50 bp, only the read length 1 (or named the first end) in each pair was used and trimmed to 50 bp for CNV analysis. In brief, an adjustable sliding window (50 kb, with 5 kb increments) was used to report candidate regions for CNV, and an adjustable non-overlapping window (5 kb) was used to identify precise boundaries by the incremental coverage ratio method. If the P value of the U test based on the population is less than 0.0001, rare CNVs are reported.

[0182] To detect AOH using GS, if consecutive windows have NRG less than 0.4 i , and 50% of these windows have an NRH greater than 1.25 i , then the region of AOH is reported. In addition, if two candidate regions (larger than 200-kb) are separated by a window (the NRG i If the AOH region was greater than 0.4 but less than 1), the two candidate regions were combined. Based on the recommendations from the International System for Human Cell Genome Nomenclature (ISCN, 2016), AOH final regions > 500 kb were reported.

[0183] By integrating NRG iSet to the average of itself and the four flanking regions (two upstream and two downstream) to obtain FNRG i , and each NRH i It was also set to the average of itself and the eight flanking regions (two upstream and two downstream) to obtain FNRH i , thus performing AOH detection of low-depth GS. If consecutive windows have FNRG less than 0.5 i , and for 25% of the windows within the candidate region, FNRH i If the value is greater than 1.25, a candidate region with AOH is reported. In addition, if there are continuous NRGs with AOH less than 0.5 in the candidate region, i The area of ​​​​the value and 25% of the windows have an NRH greater than 1.25 i In addition, if two candidate regions (larger than 200-kb) are separated by a window (the NRG i According to ISCN 2016, AOH final regions of >500 kb are also reported.

[0184] To detect AOH within mosaic trisomy events by low-depth GS, each NRM i Set to the average of itself and four flanking regions (two upstream and two downstream), giving FNRM i When the size is >1-Mb, continuous NRMs with >1.15 are reported. i area.

[0185] Determination of parental origin

[0186] For the two cases with low-depth GS of the parents, each parent in each family was tested for SNVs using the same method as the proband. Only sites where the parents were homozygous for different genotypes were selected. The number of maternal / paternal SNVs was defined as the proband with at least one allele consistent with the maternal / paternal. The ratio of maternal SNVs divided by paternal SNVs was calculated in each fixed window of size 1-Mb, and regions with extreme values ​​were reported (ratio>5 or <0.2).

[0187] Quantitative fluorescence PCR

[0188] The parental origins of chromosomes 6 and 15 reported by low-depth GS were further verified by quantitative fluorescence PCR (QF-PCR) with short tandem repeat (STR) markers selected from the UCSC genome browser following the manufacturer’s instructions as described in our previous study [ 36 ].

[0189] AOH Verification

[0190] For the trio GS data downloaded from the 1000Genomes Project, raw data (idat files) from the SNP array platform Omni 2.5M (Illumina) were downloaded from ftp: / / ftp.1000genomes.ebi.ac.uk / vol1 / ftp / release / 20130502 / supporting / hd_genotype_chip / broad_intensities / [ 37 ] and imported for AOH detection via GenomeStudio (Illumina), with default settings used for detection parameters and resolution (1-Mb) [ 5 ].

[0191] result

[0192] In this study, the sensitivity and specificity of low-depth GS for detecting AOH were first evaluated using GS as a reference and then validated using 12 clinical samples with known AOH reported by CMA.

[0193] Detection of AOH by heterozygous / homozygous SNV ratio

[0194] Since the homozygous long fragments expressed by AOH are usually equally inherited, we considered whether the similarity of the parental genomes could be detected by the ratio of heterozygous and homozygous SNVs detected. Since it is difficult to determine the similarity of the parental genotypes, the parental genome differences were used instead. The results of GS showed that the ratio of parental genome differences was positively correlated with the ratio of heterozygous SNVs ( Figure 2 a and Figure 8 a), and negatively correlated with the ratio of homozygous SNVs ( Figure 2 b and Figure 8 b). This indicates that reducing the parental genomic divergence will lead to a decrease in the rate of heterozygous SNVs and an increase in the rate of homozygous SNVs, and the rates of heterozygous and homozygous SNVs are negatively correlated ( Figure 2 c and Figure 8 c).

[0195] In addition, the results also showed that the two ratios of homozygous / heterozygous SNVs from GS were strongly correlated with the ratios from low-depth GS ( Figure 2 df and Figure 8 df), demonstrating the feasibility of using low-depth GS to detect AOH.

[0196] Evaluation of sensitivity and specificity of AOH detection

[0197] Based on the assumption that heterozygous deletions result in copy number loss AOH due to the absence of one allele, we further determined the cutoff value for the ratio of heterozygous SNVs for AOH detection in both GS and low-depth GS in eight cases with deletions larger than 500 kb from a previous study

[27] . As expected, all regions with heterozygous deletions showed a reduced ratio of heterozygous SNVs ( Fig. 9 ), however, the deviations of these ratios detected by low-depth GS were greater than those detected by GS ( Fig. 9 ). To reduce the bias of low-depth GS, we further normalized each ratio with the values ​​from the flanking windows for genome-wide screening of candidate regions with AOH and reported the precise boundaries by using the raw values.

[0198] Among these cases, we incidentally identified two AOH seq[GRCh37]2p23.2p21(29700000_42600000)x2 hmz( Fig.10 ) and seq[GRCh37]5q23q34(149200000_164900000)x2hmz( Figure 4 ), both of which were confirmed by showing a lack of parental genomic divergence in these two regions. This suggests that they may have been caused by inherited equivalence ( Fig.10 f and Figure 4 f). We further sought validation of the SNP array results at the default resolution of 1-Mb, and both AOHs were confirmed ( Fig.11 ). The reliability of the AOH test was verified by observing the reduced ratio of diploid heterozygous SNVs.

[0199] Furthermore, within these two regions, >50% and >25% of the windows had homozygous SNV ratios >1.25 in GS and low-depth GS, respectively. This result confirms that when the parental genomic divergence decreases, an increased ratio of homozygous SNVs is observed ( Figure 2 b). Therefore, we further compared the results generated by SNP array and low-depth GS by incorporating the ratio of homozygous SNVs to further exclude false positives that may be contributed by repetitive sequences (such as low copy repeats). In summary, SNP array reported 13 AOHs (>1-Mb) in 9 regions and only in HG00733, with sizes ranging from 1.1 to 8.3-Mb, while low-depth GS detected 87 AOHs (>500-kb), including 16 AOHs of 1-Mb in HG00733. All AOHs reported by SNP array were consistently detected by low-depth GS. In addition, for the other three AOHs>1-Mb reported by low-depth GS, the absence of heterozygous SNVs in these regions indicates the reliability of low-depth GS in detecting AOHs ( Fig.12 ).

[0200] Overall, by incorporating filtering for both diploid heterozygous and homozygous SNV ratios, we achieved 100% sensitivity and specificity for detecting AOH by low-depth GS when the resolution was set to 1.4-Mb using GS results as a reference. Figure 3 It is noteworthy that when the resolution was set to 1-Mb, both the sensitivity and specificity reached >90.0% ( Figure 3 b).

[0201] Validation with clinical samples known to have AOH

[0202] We further applied low-depth GS to 12 clinical samples (from 10 cases) known to have multiple AOH reported by CMA (Table 1). After detection of AOH, the results showed 100% concordance with the results reported by CMA (resolution cutoff was set at 5-Mb [5], Table 1). In addition, due to the lack of sufficient SNP probes in the target region of the CMA platform, low-depth GS was able to report additional cryptic AOH ( Figure 4 ). Among these cases, low-depth GS reported 4 cases of AOH affecting multiple chromosomes and 8 cases affecting a single chromosome (Table 1). In addition, to evaluate the reproducibility of this method, the complete experiment was repeated for 5 of the 12 samples, including library construction, sequencing, and data analysis. Under the same parameters, when the resolution was set to 1.0-Mb, 100% concordance was achieved between the data from the first batch and the replicate data ( Figure 3 ef).

[0203] In addition, an increased rate of “mosaic” heterozygous SNVs and the co-occurrence of mosaic trisomies across the affected chromosome were observed in both CVS samples ( Figure 6 and Fig.13 This indicates a biased fraction of one allele across the entire affected chromosome, consistent with observations from CMA ( Figure 6 b). Among them, case ( Figure 6 ) had chorionic villus sample 18C1493, amniotic fluid (18C1564) and fetal umbilical cord blood sample (aCGH15274). Compared with the results of chorionic villus, amniotic fluid and fetal umbilical cord blood samples showed diploid chromosome 6, but with multiple AOH ( Figure 5 ), which may be caused by UPD[9]. To confirm the existence of uniparental heterodisomy, we further performed low-depth GS on the parents. The results of AF and fetal umbilical cord blood samples showed that the diploid chromosome 6 pair was of maternal origin ( Figure 6g), and then confirmed by QF-PCR with STR markers. Thus, the complete study of multiple sample types in this family confirmed the case of trisomy rescue and also demonstrated the reliability of AOH detection by low-depth GS. In addition, the ability to determine the maternal origin of each chromosome / segment by low-depth GS was further confirmed by confirming the uniparental disomy of chromosome 15, which was maternal in sample 16C0067.

[0204] In addition, the advantages of applying low-depth GS in identifying precise boundaries and reporting cryptic AOH were further demonstrated in consanguineous families (Table 1). As the brother of 17C1122, 17C1176, was diagnosed with myoclonic seizures, developmental delay, dysarthria, and truncal ataxia, he submitted chorionic villus sampling for prenatal diagnosis at 12+2 gestational weeks. ES in his brother identified a homozygous variant NM_153033:c50T>A in KCTD7, causing autosomal recessive progressive myoclonic epilepsy-3 (EPM3, OMIM: 611726) with / without intracellular inclusions, while this variant was heterozygous in the unaffected sibling 17C1175. Both low-depth GS and CMA detected an AOH region seq[GRCh37]7q11.21q11.23(65500000_72400000)x2 hmz in 17C1176, covering KCTD7, which was absent in 17Cl175 ( Figure 7 Although CMA did not report AOH involving KCTD7 in the fetus, Sanger sequencing at the time of prenatal diagnosis confirmed the presence of a homozygous wild-type T in 17C1122. The pregnancy continued and ultimately resulted in a normal live birth. By blind testing, low-depth GS reported an additional 1.2-Mb AOH involving KCTD7 in 17C1122 seq[GRCh37]7q11.21(65500000_66700000)x2 hmz( Figure 7), confirming that the presence of homozygous wild-type T in 17C1122 was caused by this small AOH. We further discussed whether genotype information was identified in these three cases, 17C1122, 17C1175 and 17C1176, respectively. Homozygous wild-type T with 6 supporting reads was detected in 17C1122; heterozygous base type AT with three reads was reported in 17C1175, and homozygous mutant type A with seven reads was reported in 17C1176. All base calls were consistent with previous Sanger sequencing results, confirming the possibility of identifying pathogenic mutations with accurate genotypes (heterozygous / homozygous) by low-depth GS, although the read depth coverage is not as high as GS. This was further confirmed by showing that the lack of hemizygous TCA haplotypes (average of three supporting reads due to the lack of one copy in this region) in low-depth GS is consistent with the results provided by GS

[27] .

[0205] Overall, this study shows the robustness of low-depth GS in detecting AOH at significantly higher resolution, with precise boundaries detected and discrimination between uniparental heterodisomy and homodisomy.

[0206] discuss

[0207] In this study, we describe a robust, platform-neutral method for identifying genome-wide absence of heterozygosity (AOH) by low depth (GS, ~4x sequencing depth). By comparing with GS (>30x sequencing depth) from 53 cases, our study showed that the sensitivity and specificity of detecting AOH with low-depth GS reached >90.0% at a resolution of 1-Mb and 100% at a resolution of 1.4-Mb. In addition, in 12 clinical samples with reported AOH, this method not only confirmed all known AOH and reported uniparental heterodisomy and homodisomy, but also detected additional cryptic AOH, providing accurate genotypes. In the replication study, 100% concordance was achieved between the data from the first batch and the replicated data when the resolution was set to 1.0-Mb. In summary, this study demonstrated the robustness and reproducibility of this method in AOH detection.

[0208] In this study, the ratio of heterozygous and homozygous SNVs was mainly used to detect AOH. Supporting observations were that parental genomic differences were positively correlated with the ratio of heterozygous SNVs and negatively correlated with the ratio of homozygous SNVs. In addition, the high correlation of the ratio of heterozygous / homozygous SNVs between GS and low-depth GS demonstrated the reliability of using low-depth GS for detection ( Figure 2In addition, our method not only reported 13 AOHs (>1-Mb) that were consistent with the high-density SNP array (with a total of 2.5 million probes), but also detected three cryptic novel AOHs in the highly referenced case HG00733 [6, 26] ( Fig.12 Among the total 16 AOHs reported by low-depth GS, there are two AOHs with lengths greater than 10-Mb ( Figure 4 and Fig.11 ). Within these two regions, 7 and 23 OMIM pathogenic genes were reported, respectively, including autosomal dominant genes (i.e., SOS1) and recessive genes (i.e., CYP1B1). Although these two AOH were reported in presumed normal individuals, the involvement of OMIM pathogenic genes emphasizes the importance of AOH detection, and this finding demonstrates the importance of defining AOH profiles by using data from presumed normal individuals (e.g., from the 1000 Genomes Project).

[0209] We further confirmed 100% concordance of AOH detection between low-depth GS and conventional CMA (5-Mb, the maximum resolution of the CMA platform used) using 12 clinical samples with multiple AOH reported by CMA. In addition, the importance of detecting AOH at a higher resolution was confirmed by identifying a cryptic 1.2-Mb AOH in a prenatal case involving KCTD7, where homozygous variants in KCTD7 caused severe phenotypes in older siblings due to the presence of large fragments of AOH. In addition, low-depth GS also showed the possibility of providing accurate genotypes in this family (fetus and two older siblings) and in hemizygous alleles of the recurrent deletion syndrome at 16p11.22, although the number of supporting reads was limited. Based on this increased resolution, we were able to identify those key regions known to carry imprinted genes, such as the 2-Mb domain on chromosome 15q11-q13 that affects Prade-willi and Angelman syndromes

[40] . In addition, for two cases with low-depth GS results for both parents, we demonstrated the feasibility of determining parental origin using genotype information supported by limited read depth. Using this information, we were able to identify the parental origin in the presence of uniparental homodisomy (AOH, Figure 5 g) Identification of uniparental heterodisomy in the affected chromosome (absence of AOH).

[0210] The method is sequencing platform neutral (applicable to data generated from Illumina and MGI) and is independent of sequencing read length (126 bp in data downloaded from the 1000 Genomes Project and 100 bp in data sequenced in this study), offering the possibility of integrating the test into sequencing runs of ES or GS. Currently, many laboratories offer GS / ES tests with paired-end 150 bp sequencing, and the number of read pairs required to achieve the approximately 4-fold sequencing depth required for AOH analysis can be set as low as 4 million, suggesting that this would be one of the most cost-effective tests.

[0211] Overall, this study shows the reliability of combining the ratios of "germline" / "mosaic" heterozygous and homozygous SNVs for the identification of germline and mosaic AOH. For example, a combination of reduced "germline" heterozygous SNV ratios and increased homozygous SNV ratios was used to identify AOH. In addition, the combination of different parameters will aid in CNV detection. For example, all ratios are reduced due to heterozygous deletions, or increased ratios of "mosaic" heterozygous SNVs in regions with duplications.

[0212] in conclusion

[0213] This study describes a robust method to detect AOH by utilizing low-depth GS (approximately 4 times the sequencing depth) at significantly higher resolution than conventional CMA and even high-density SNP arrays. Furthermore, by showing significantly high concordance of low-depth GS in the detection of AOH compared with results reported by GS and CMA, our study provides strong evidence for implementation of this method in the setting of routine genetic testing using low-depth GS.

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Claims

1. A method for detecting absence of heterozygosity (AOH) in a biological sample from an individual for non-diagnostic purposes, the method comprising: (i) receiving low-depth sequence reads of genomic DNA from the biological sample, wherein the low-depth genomic sequencing has a sequencing depth of 3-5 times; (ii) aligning the sequence reads to a human genome reference, and selecting and sorting the sequence reads aligned to the human genome reference based on the aligned chromosome and genome coordinates; (iii) identifying single nucleotide variations (SNVs) in the aligned sequence reads, wherein the SNV at each site has a mutant base type that is different from the base type at the corresponding site of the human genome reference; (iv) identifying a homozygous SNV, a diploid heterozygous SNV, or a non-diploid heterozygous SNV from the SNVs identified in step (iii), wherein A homozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference, A diploid heterozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being no less than 25% and no more than 75%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference, defining a non-diploid heterozygous SNV based on a percentage of sequence reads supporting a mutant base type being less than 25% and greater than 0% or greater than 75% and less than 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference; (v) determining, for the window, a ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV identified in step (iv), wherein the ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV represents the ratio of the number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs of the window to the average number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs among all windows in the biological sample; and (vi) comparing the ratio of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs of each window determined by step (v) with the average ratio of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs of the corresponding respective windows established from a control population, wherein a decreased ratio of diploid heterozygous SNVs and an increased ratio of homozygous SNVs indicates AOH, or an increased ratio of non-diploid heterozygous SNVs indicates mosaic AOH, The biological sample is selected from the group consisting of peripheral blood, chorionic villi, amniotic fluid, umbilical cord blood and tissue samples from organs.

2. The method of claim 1, wherein the biological sample is from placental tissue.

3. The method of claim 1, wherein the individual is a pregnant woman, an infant, an individual suffering from cancer, or an individual suspected of suffering from cancer.

4. The method of claim 1, wherein the sequence read length is a single-end sequence read length or a double-end sequence read length.

5. The method of claim 1, wherein the low-depth genome sequencing has a sequencing depth of 3. The method of claim 1 , wherein the human genome reference is GRCh37 / hg19 or GRCh38 / hg38.

7. The method of claim 1, wherein the alignment step is performed using Short Oligonucleotide Alignment Program 2 (SOAP2) or Burrows-Wheeler Alignment Program (BWA) and Bowtie2.

8. The method of claim 1, wherein step (ii) further comprises removing sequence reads resulting from polymerase chain reaction (PCR) duplications.

9. The method of claim 1, wherein step (iii) further comprises deleting the site described below: (a) The minimum read depth of a locus is determined by the minimum read depth of the biological sample; (b) the maximum read depth of the locus is determined by the maximum read depth of the biological sample; or (c) Sites where no sequence reads supported the mutant base type.

10. The method of claim 1, wherein the window in step (v) has a fixed length.

11. The method of claim 10, wherein the window in step (v) has a length of 100 kb.

12. The method of claim 1, wherein step (v) comprises: determining the number of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs of the window, determining the average number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs in all windows in the biological sample, and The ratio of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs for the window is calculated by dividing the number of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs identified for the window by the average number of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs for all windows in the biological sample.

13. The method of claim 1, wherein the control population is of the same gender as the individual.

14. The method of claim 13, wherein the control population has at least 30 control individuals.

15. The method of claim 1, wherein the loss of heterozygosity is copy number neutral loss of heterozygosity (CN-LOH).

16. The method of claim 1, wherein step (vi) comprises: The ratio of the homozygous SNV, diploid heterozygous SNV or non-diploid heterozygous SNV of the window is normalized relative to the average ratio of the homozygous SNV, diploid heterozygous SNV or non-diploid heterozygous SNV of the corresponding window established from the control population, thereby providing the corresponding ratio ratio of the homozygous SNV, diploid heterozygous SNV or non-diploid heterozygous SNV of the window.

17. The method of claim 16, wherein step (vi) further comprises: In the case where copy number neutrality is represented as a copy ratio equal to 1, for all windows with a diploid heterozygous SNV ratio ratio less than 1, if there are consecutive windows with a diploid heterozygous SNV ratio ratio less than 0.5 and the percentage of windows with a homozygous ratio ratio greater than 1.25 is at least 30%, then these windows are defined as one region, or if there is no more than one window with a diploid heterozygous SNV ratio ratio greater than 0.5 but less than 1, then two regions are combined into one region; and The area is reported as having AOH present.

18. The method of claim 16, wherein step (vi) further comprises: In the case of copy number mosaic repeats represented by copy ratios greater than 1, for all windows with a non-diploid heterozygous SNV ratio ratio greater than 1, if there are consecutive windows with a non-diploid heterozygous SNV ratio ratio greater than 1.15, these windows are defined as a region; and Such regions are reported as having chimeric AOH.

19. The method of claim 1, wherein the average ratio of heterozygous SNVs for corresponding individual windows established from a control population is determined by: (ci) receiving low-depth sequence reads of genomic DNA from a biological sample of a control individual from a control population; (cii) aligning the sequence reads with a human genome reference, and selecting and sorting the sequence reads aligned with the human genome reference based on the aligned chromosome and genome coordinates; (ciii) identifying single nucleotide variations (SNVs) in the aligned sequence reads, wherein the SNV at each site has a mutant base type that is different from the base type at the corresponding site in the human genome reference; (civ) identifying a homozygous SNV, a diploid heterozygous SNV, or a non-diploid heterozygous SNV from the SNVs identified in step (ciii), wherein A homozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference, A diploid heterozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being no less than 25% and no more than 75%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference, defining a non-diploid heterozygous SNV based on a percentage of sequence reads supporting a mutant base type being less than 25% and greater than 0% or greater than 75% and less than 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference; (cv) determining, for the window, a ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV identified in step (civ), wherein the ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV represents the ratio of the number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs of the window to the average number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs among all windows in the biological sample from the control individual; and (cvi) averaging the ratios of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs for the windows from all control individuals to provide an average ratio of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs for the corresponding windows of the control population.

20. The method of claim 19, further comprising a sex determination step performed between steps (cii) and (ciii), wherein the alignment ratios of the X chromosome, the Y chromosome and the whole genome are calculated as the number of sequence reads aligned to the chromosome / genome respectively divided by the length defined by the human reference genome, the Y chromosome percentage is calculated as the alignment ratio of the Y chromosome divided by the alignment ratio of the whole genome, and if the Y chromosome percentage is greater than 0.1, the control individual is considered to be male.

21. The method of claim 20, wherein steps (ciii) to (cvi) are performed separately on male and female control individuals based on the results of the gender determination step.

22. A method as claimed in claim 19, wherein in step (cvi), if the ratio of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs in a window between control individuals has a significant deviation, then the average ratio of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs in the window is calculated as the average of the ratios of the window and its flanking windows.

23. The method of claim 22, wherein the flanking windows are two upstream windows and two downstream windows.

24. A computer system for detecting absence of heterozygosity (AOH) in a biological sample from an individual, the computer system comprising a processor and a memory storing a plurality of instructions, wherein the processor, when processing the instructions, is configured to: (i) receiving low-depth sequence reads of genomic DNA from the biological sample, wherein the low-depth genomic sequencing has a sequencing depth of 3-5 times; (ii) aligning the sequence reads to a human genome reference, and selecting and sorting the sequence reads aligned to the human genome reference based on the aligned chromosome and genome coordinates; (iii) identifying single nucleotide variations (SNVs) in the aligned sequence reads, wherein the SNV at each site has a mutant base type that is different from the base type at the corresponding site of the human genome reference; (iv) identifying a homozygous SNV, a diploid heterozygous SNV, or a non-diploid heterozygous SNV from the SNVs identified in (iii), wherein A homozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference, A diploid heterozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being no less than 25% and no more than 75%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference, defining a non-diploid heterozygous SNV based on a percentage of sequence reads supporting a mutant base type being less than 25% and greater than 0% or greater than 75% and less than 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference; (v) determining, for the window, a ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV identified in (iv), wherein the ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV represents the ratio of the number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs of the window to the average number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs among all windows in the biological sample; and (vi) comparing the ratio of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs of each window determined by (v) with the average ratio of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs of the corresponding respective windows established from a control population, wherein a decreased ratio of diploid heterozygous SNVs and an increased ratio of homozygous SNVs indicates AOH, or an increased ratio of non-diploid heterozygous SNVs indicates mosaic AOH, The biological sample is selected from the group consisting of peripheral blood, chorionic villi, amniotic fluid, umbilical cord blood and tissue samples from organs.

25. The computer system of claim 24, wherein the biological sample is placental tissue.

26. The computer system of claim 24, wherein the individual is a pregnant woman, an infant, an individual suffering from cancer, or an individual suspected of suffering from cancer.

27. The computer system of claim 24, wherein the sequence read is a single-end sequence read or a double-end sequence read.

28. The computer system of claim 24, wherein the low-depth genome sequencing has a sequencing depth of 3 times.

29. The computer system of claim 24, wherein the human genome reference is GRCh37 / hg19 or GRCh38 / hg38.

30. The computer system of claim 24, wherein the alignment operation is performed using Short Oligonucleotide Alignment Program 2 (SOAP2) or Burrows-Wheeler Alignment Program (BWA) and Bowtie2.

31. The computer system of claim 24, wherein the processor, when processing the instructions, is further configured to remove sequence read lengths resulting from polymerase chain reaction (PCR) duplications.

32. The computer system of claim 24, wherein the processor, when processing the instruction, is further configured to delete the location described below: (a) The minimum read depth of a locus is determined by the minimum read depth of the biological sample; (b) the maximum read depth of the locus is determined by the maximum read depth of the biological sample; or (c) Sites where no reads support the mutant base type.

33. The computer system of claim 24, wherein the window has a fixed length.

34. The computer system of claim 33, wherein the window has a length of 100 kb.

35. The computer system of claim 24, wherein the processor, when processing the instruction, is further configured to: determining the number of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs of the window, determining the average number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs in all windows in the biological sample, and The ratio of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs for the window is calculated by dividing the number of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs identified for the window by the average number of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs for all windows in the biological sample.

36. The computer system of claim 24, wherein the control population has the same gender as the individual.

37. The computer system of claim 36, wherein the control population has at least 30 control individuals.

38. The computer system of claim 24, wherein the loss of heterozygosity is copy number neutral loss of heterozygosity (CN-LOH).

39. The computer system of claim 24, wherein the processor, when processing the instruction, is further configured to: The ratio of the homozygous SNV, diploid heterozygous SNV or non-diploid heterozygous SNV of the window is normalized relative to the average ratio of the homozygous SNV, diploid heterozygous SNV or non-diploid heterozygous SNV of the corresponding window established from the control population, thereby providing the corresponding ratio ratio of the homozygous SNV, diploid heterozygous SNV or non-diploid heterozygous SNV of the window.

40. The computer system of claim 39, wherein the processor, when processing the instruction, is further configured to: In the case where copy number neutrality is represented as a copy ratio equal to 1, for all windows with a diploid heterozygous SNV ratio ratio less than 1, if there are consecutive windows with a diploid heterozygous SNV ratio ratio less than 0.5 and the percentage of windows with a homozygous ratio ratio greater than 1.25 is at least 30%, then these windows are defined as one region, or if there is no more than one window with a diploid heterozygous SNV ratio ratio greater than 0.5 but less than 1, then two regions are combined into one region; and The area is reported as having AOH present.

41. The computer system of claim 39, wherein the processor, when processing the instruction, is further configured to: In the case of copy number mosaic duplication represented as a copy ratio greater than 1 or copy number neutrality represented as a copy ratio equal to 1, for all windows with a non-diploid heterozygous SNV ratio ratio greater than 1, if there are consecutive windows with a non-diploid heterozygous SNV ratio ratio greater than 1.15, define these windows as a region; and Such regions are reported as having chimeric AOH.

42. A computer readable medium storing a plurality of instructions, wherein the plurality of instructions, when executed by one or more processors, perform operations comprising: (i) receiving low-depth sequence reads of genomic DNA from a biological sample of an individual, wherein the low-depth genomic sequencing has a sequencing depth of 3-5 times; (ii) aligning the sequence reads to a human genome reference, and selecting and sorting the sequence reads aligned to the human genome reference based on the aligned chromosome and genome coordinates; (iii) identifying single nucleotide variations (SNVs) in the aligned sequence reads, wherein the SNV at each site has a mutant base type that is different from the base type at the corresponding site of the human genome reference; (iv) identifying a homozygous SNV, a diploid heterozygous SNV, or a non-diploid heterozygous SNV from the SNVs identified in (iii), wherein A homozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference, A diploid heterozygous SNV is defined based on a percentage of sequence reads supporting a mutant base type being no less than 25% and no more than 75%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference, defining a non-diploid heterozygous SNV based on a percentage of sequence reads supporting a mutant base type being less than 25% and greater than 0% or greater than 75% and less than 100%, wherein the mutant base type is different from the base type at the corresponding site of the human genome reference; (v) determining, for the window, a ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV identified in (iv), wherein the ratio of the homozygous SNV, diploid heterozygous SNV, or non-diploid heterozygous SNV represents the ratio of the number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs of the window to the average number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs among all windows in the biological sample; and (vi) comparing the ratio of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs of each window determined by (v) with the average ratio of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs of the corresponding respective windows established from a control population, wherein a decreased ratio of diploid heterozygous SNVs and an increased ratio of homozygous SNVs indicates AOH, or an increased ratio of non-diploid heterozygous SNVs indicates mosaic AOH, The biological sample is selected from the group consisting of peripheral blood, chorionic villi, amniotic fluid, umbilical cord blood and tissue samples from organs.

43. The computer readable medium of claim 42, wherein the biological sample is a placenta.

44. The computer-readable medium of claim 42, wherein the individual is a pregnant female, an infant, an individual having cancer, or an individual suspected of having cancer.

45. The computer-readable medium of claim 42, wherein the sequence reads are single-end sequence reads or double-end sequence reads.

46. ​​The computer-readable medium of claim 42, wherein the low-depth genome sequencing has a sequencing depth of 3 times.

47. The computer-readable medium of claim 42, wherein the human genome reference is GRCh37 / hg19 or GRCh38 / hg38.

48. The computer readable medium of claim 42, wherein the alignment is performed using Short Oligonucleotide Alignment Program 2 (SOAP2) or Burrows-Wheeler Alignment Program (BWA) and Bowtie2.

49. The computer-readable medium of claim 42, wherein (ii) further comprises removing sequence read lengths due to polymerase chain reaction (PCR) duplications.

50. The computer-readable medium of claim 42, wherein (iii) further comprises deleting the site described below: (a) The minimum read depth of a locus is determined by the minimum read depth of the biological sample; (b) the maximum read depth of the locus is determined by the maximum read depth of the biological sample; or (c) Sites where no reads support the mutant base type.

51. The computer-readable medium of claim 42, wherein the window in (iv) has a fixed length.

52. The computer readable medium of claim 51, wherein the window has a length of 100 kb.

53. The computer-readable medium of claim 42, wherein (v) comprises: determining the number of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs of the window, determining the average number of homozygous SNVs, diploid heterozygous SNVs, or non-diploid heterozygous SNVs in all windows in the biological sample, and The ratio of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs for the window is calculated by dividing the number of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs identified for the window by the average number of homozygous SNVs, diploid heterozygous SNVs or non-diploid heterozygous SNVs for all windows in the biological sample.

54. The computer-readable medium of claim 42, wherein the control population has the same gender as the individual.

55. The computer readable medium of claim 54, wherein the control population has at least 30 control individuals.

56. The computer readable medium of claim 42, wherein step (vi) comprises: The ratio of the homozygous SNV, diploid heterozygous SNV or non-diploid heterozygous SNV of the window is normalized relative to the average ratio of the homozygous SNV, diploid heterozygous SNV or non-diploid heterozygous SNV of the corresponding window established from the control population, thereby providing the corresponding ratio ratio of the homozygous SNV, diploid heterozygous SNV or non-diploid heterozygous SNV of the window.

57. The computer-readable medium of claim 56, wherein (vi) further comprises: In the case where copy number neutrality is represented as a copy ratio equal to 1, for all windows with a diploid heterozygous SNV ratio ratio less than 1, if there are consecutive windows with a diploid heterozygous SNV ratio ratio less than 0.5 and the percentage of windows with a homozygous ratio ratio greater than 1.25 is at least 30%, then these windows are defined as one region, or if there is no more than one window with a diploid heterozygous SNV ratio ratio greater than 0.5 but less than 1, then two regions are combined into one region; and The area is reported as having AOH present.

58. The computer-readable medium of claim 56, wherein (vi) further comprises: In the case of copy number mosaic duplication represented as a copy ratio greater than 1 or copy number neutrality represented as a copy ratio equal to 1, for all windows with a non-diploid heterozygous SNV ratio ratio greater than 1, if there are consecutive windows with a non-diploid heterozygous SNV ratio ratio greater than 1.15, define these windows as a region; and Such regions are reported as having chimeric AOH.

59. An apparatus comprising one or more processors and a computer readable medium as claimed in any one of claims 42 to 58.

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