A method, device, system and storage medium for analyzing contamination of sequencing samples

Abstract

The present invention provides an analysis method, device, system and storage medium for sequencing sample contamination, and relates to the technical field of high-throughput sequencing. The analysis method comprises: determining the SNP sites of the target sample and obtaining the mutation frequency corresponding to each SNP site; classifying the SNP sites according to the mutation frequency to obtain the site typing category and the statistical number; determining the category and statistical number of abnormal sites in the target sample; and obtaining the contamination analysis result according to the statistical number. The present invention introduces SNP sites into the gene detection panel, judges the suspected contaminated samples by the abnormal frequency, thereby analyzing whether the samples are contaminated and obtaining the contamination analysis result. For samples in the high-throughput sequencing process, on the one hand, it provides an effective way for the analysis and detection of sample contamination, and on the other hand, it can more effectively identify and exclude false positive results, thereby ensuring the accuracy of high-throughput sequencing data and the authenticity of experimental results.

Description

Technical Field

[0001] The present invention relates to the field of high-throughput sequencing technology, and more specifically, to an analysis method, device, system and storage medium for sequencing sample contamination. Background Art

[0002] With the rapid development of biotechnology, the second-generation gene sequencing technology (Next-Generation Sequencing, NGS) has become an important tool for genetic research. This technology can read a large amount of genetic information efficiently and quickly, thereby supporting various biological studies, including the study of the pathological mechanisms of complex diseases, the study of genetic diversity, and the realization of personalized medicine. NGS technology significantly improves sequencing efficiency and reduces costs by processing large-scale samples in parallel, thus promoting a new round of revolution in biomedical research.

[0003] However, in actual operation, the second-generation gene sequencing technology faces many challenges, especially the problem of sample cross-contamination is becoming increasingly prominent. In the process of high-throughput sequencing, due to the need to process a large number of samples, there are multiple links that may cause cross-contamination. For example, foreign DNA may be introduced during the DNA extraction process; cross-contamination may be generated through aerosols during the PCR process; experimental operators may forget to change the gun tip when processing samples, or when using paraffin-embedded tumor tissue sample sections, they directly proceed to cut the next sample without changing the blade. In addition, sample index misassignment (index hopping) is also a common problem in high-throughput sequencing, further increasing the risk of contamination.

[0004] These contamination issues are particularly serious in studies that require the detection of low-frequency mutations. For example, in tumor gene testing projects, even minor contamination can lead to false positive results, that is, the detection of mutations that are not present in the sample. This not only affects the accuracy of the experimental results, but may also lead to errors in clinical diagnosis and treatment decisions. At present, although the occurrence of contamination can be reduced to a certain extent through standardized operations, it is still a challenge to completely avoid cross-contamination due to the complexity of the operating procedures and the large sample size.

[0005] In summary, although the second-generation gene sequencing technology has shown great potential and advantages in many fields, the existence of sample contamination has seriously affected the reliability of its results and the breadth of its application. In the genetic testing of important diseases such as tumors, false positive mutations caused by contamination are particularly likely to have serious consequences. Therefore, it is urgent to develop new technologies or improve existing technologies to more effectively identify and exclude these false positive results and ensure the accuracy of high-throughput sequencing data and the authenticity of experimental results. Summary of the invention

[0006] In view of this, in order to solve the above technical problems, the present invention provides a method for analyzing sequencing sample contamination, comprising:

[0007] Determine the SNP site of the target sample, and obtain the mutation frequency corresponding to each SNP site of the target sample;

[0008] Classify the SNP sites of the target sample according to the mutation frequency to obtain the site typing category corresponding to each SNP site and the statistical number corresponding to each site typing category; wherein the site typing category includes wild-type sites, homozygous mutant sites and heterozygous mutant sites;

[0009] Based on the mutation frequency, determine the abnormal site category in the target sample and the statistical number corresponding to each abnormal site category; wherein the abnormal site category includes WT contaminated sites and Hom contaminated sites;

[0010] According to the statistical numbers corresponding to the site typing categories and the abnormal site categories, the contamination analysis results of the SNP sites are obtained.

[0011] Preferably, obtaining the contamination analysis result of the SNP site according to the statistical number corresponding to the site typing category and the abnormal site category includes:

[0012] Calculate the abnormality rate of the target sample according to the statistical number of the site typing category and the abnormal site category;

[0013] Determining whether the abnormality rate of the target sample is no greater than 10%;

[0014] If the abnormality rate of the target sample is not greater than 10%, the contamination analysis result is determined to be that the target sample is not contaminated;

[0015] If the abnormality rate of the target sample is greater than 10%, the contamination analysis result is determined to be that the target sample is contaminated.

[0016] Preferably, in the site typing category, the mutation frequency corresponding to the wild-type site is 0%-1%; the mutation frequency corresponding to the heterozygous mutant site is 30%-70%; the mutation frequency corresponding to the homozygous mutant site is 99%-100%;

[0017] In the abnormal site category, the mutation frequency corresponding to the WT contamination site is 1%-10%; the mutation frequency corresponding to the Hom contamination site is 90%-99%;

[0018] The calculation method of the abnormality rate is:

[0019]

[0020] Among them, ratio represents the abnormal rate; YC1 represents the statistical number of the WT contaminated sites; YC2 represents the statistical number of the Hom contaminated sites; WT represents the statistical number of the wild-type sites; Hom represents the statistical number of the homozygous mutant sites.

[0021] Preferably, after determining that the contamination analysis result is that the target sample has been contaminated, the method further includes:

[0022] Calculating the mean mutation frequency of the SNP sites with a mutation frequency in the range of 1%-10% in the target sample as a first mean; and calculating the mean mutation frequency of the SNP sites with a mutation frequency in the range of 90%-99% in the target sample as a second mean;

[0023] A numerical range of the predicted contamination ratio corresponding to the target sample is determined according to the first mean and the second mean.

[0024] Preferably, determining the numerical range of the predicted contamination ratio corresponding to the target sample according to the first mean and the second mean includes:

[0025] The maximum and minimum extreme values ​​in the predicted pollution ratio are determined according to the following formula:

[0026]

[0027] C Het =C Hom ×2;

[0028] The numerical range of the predicted contamination ratio of the target sample is: C Ho… ≤C 预测 ≤C Het ;

[0029] Among them, C Hom The contamination ratio when all SNP sites representing the contamination source are homozygous mutant sites is taken as the minimum extreme value; C Het The contamination ratio when all SNP sites representing the contamination source are heterozygous mutant sites is taken as the maximum extreme value; represents the first mean; represents the second mean; C 预测 Represents the predicted contamination ratio of the target sample.

[0030] Preferably, after determining that the contamination analysis result is that the target sample has been contaminated, the method further includes:

[0031] If the abnormality rate of the target sample is greater than 10%, a preset abnormality rate expected value-pollution source sample number correspondence relationship is obtained, and the abnormality rate of the target sample is compared with the abnormality rate expected value in the preset abnormality rate expected value-pollution source sample number correspondence relationship; wherein the preset abnormality rate expected value-pollution source sample number correspondence relationship includes the abnormality rate expected value and the predicted pollution source sample number corresponding to the abnormality rate expected value;

[0032] If the abnormality rate is equal to any one of the abnormality rate expected values ​​in the preset abnormality rate expected value-pollution source sample number correspondence relationship, the predicted pollution source sample number corresponding to the abnormality rate expected value is used as the pollution source number of the target sample;

[0033] If the abnormality rate is not equal to any of the WT-Hom site expected values ​​in the preset abnormality rate expected value-contamination source sample number correspondence, then the two abnormality rate expected values ​​before and after which are numerically similar in the abnormality rate expected value are taken as the two end values, and the two predicted contamination source sample numbers corresponding to the two end values ​​are taken as the numerical range of the number of contamination sources of the target sample.

[0034] Preferably, the method for establishing the correspondence between the preset abnormality rate expected value and the number of pollution source samples includes:

[0035] Establish an uncontaminated sample group, determine the SNP sites that are the same as the target sample, and obtain the expected total number of wild-type sites and homozygous mutant sites of the uncontaminated samples in the uncontaminated sample group, as well as the population frequency of the wild-type allele and the population frequency of the mutant allele corresponding to the SNP site of each uncontaminated sample in the uncontaminated sample group; wherein the calculation method of the total expected number is: based on the SNP site, obtain the number of WT+Hom sites of each uncontaminated sample; calculate the mean number of the WT+Hom sites in the uncontaminated sample group as the total expected number;

[0036] According to the population frequency of the wild-type allele and the population frequency of the mutant allele, the comprehensive probability of each SNP site producing an abnormal frequency after being contaminated by contamination sources with different predicted numbers of contamination source samples is calculated;

[0037] For each number of samples of the predicted pollution source, the comprehensive probability of all SNPs producing abnormal frequencies is summed up to obtain the expected value of the number of abnormal sites produced by the uncontaminated sample after it is assumed to be contaminated;

[0038] The expected value of the abnormality rate of the uncontaminated sample after being assumed to be contaminated is calculated according to the expected value of the number of abnormal sites and the expected value of the total number of wild-type sites and homozygous mutant sites in the uncontaminated sample;

[0039] According to different predicted numbers of pollution source samples and the expected value of the abnormality rate corresponding to the predicted number of pollution source samples, a corresponding relationship between the preset expected value of abnormality rate and the number of pollution source samples is constructed.

[0040] Preferably, the comprehensive probability of abnormal frequency of each SNP site after being contaminated by pollution sources with different predicted pollution source sample numbers is calculated based on the population frequency of the wild-type allele and the population frequency of the mutant allele, including:

[0041] When the number of samples of the predicted pollution source is 1, the comprehensive probability of abnormal frequency after being polluted by the pollution source is calculated according to the following formula:

[0042] Prob1 = 2 × p × q × (1-p × q);

[0043] When the number of samples of the predicted pollution source is greater than 1, the comprehensive probability of abnormal frequency after being polluted by the pollution source is calculated according to the following formula:

[0044] Prob n =1-(p 2(n+1) +2pq+q 2(n+1) );

[0045] Where n represents the number of samples of the predicted pollution source; Prob n It represents the comprehensive probability of abnormal frequency after being contaminated by the pollution source when the number of samples of the pollution source is predicted to be n; p represents the population frequency of the wild-type allele; q represents the population frequency of the mutant allele.

[0046] Preferably, the sum of the comprehensive probabilities of abnormal frequencies corresponding to each number of samples of the predicted pollution source is added up to obtain the expected value of the number of abnormal sites generated by the uncontaminated sample after it is assumed to be contaminated, including:

[0047] The expected value of the number of abnormal sites is calculated by the following expression:

[0048]

[0049] Among them, S 总 represents the expected value of the number of abnormal sites; i represents the number of the SNP site; Prob i It represents the comprehensive probability of abnormal frequency after being contaminated by the pollution source when the SNP site is i; N represents the total number of SNP sites.

[0050] Preferably, the expected value of the abnormal rate corresponding to the uncontaminated sample is calculated based on the expected value of the number of abnormal sites and the total number of wild-type sites and homozygous mutant sites in the uncontaminated sample, including:

[0051] The expected value of the abnormal rate is calculated by the following formula:

[0052]

[0053] Among them, ratio 期望 represents the expected value of the abnormal rate of the uncontaminated sample after it is assumed to be contaminated; S 总 represents the expected value of the number of abnormal sites; N WT+Hom Represents the expected total number of wild-type sites and homozygous mutant sites in the uncontaminated sample.

[0054] Preferably, if the abnormality rate is equal to any one of the abnormality rate expected values ​​in the preset abnormality rate expected value-pollution source sample number correspondence relationship, then after taking the predicted pollution source sample number corresponding to the abnormality rate expected value as the pollution source number of the target sample, the method further includes:

[0055] If the number of the pollution sources is equal to 1, then obtaining the sample to be screened and the mutation frequency corresponding to each of the SNP sites in the sample to be screened;

[0056] The mutation frequencies of the SNP sites of the target sample and the sample to be screened are grouped to obtain a first group, a second group, a third group and a fourth group, respectively; wherein the mutation frequency of the first group ranges from 0% to 1%; the mutation frequency of the second group ranges from 1% to 10%; the mutation frequency of the third group ranges from 90% to 99%; and the mutation frequency of the fourth group ranges from 99% to 100%;

[0057] Determine the types of contamination sites corresponding to the first group, the second group, the third group, and the fourth group in the target sample; the types of contamination sites include wild-type sites, homozygous mutant sites, and heterozygous mutant sites;

[0058] According to the contamination site type and the mutation frequencies of the first group, the second group, the third group and the fourth group of the samples to be screened, the contamination source results corresponding to the samples to be screened are obtained.

[0059] Preferably, in the target samples, the types of contamination sites corresponding to the first group, the second group, the third group and the fourth group include:

[0060] In the target sample, the type of the contamination site in the contamination source sample of the site in the first group is the wild-type site;

[0061] In the target sample, the type of the contamination site in the contamination source sample of the site in the second group is the heterozygous mutant site or the homozygous mutant site;

[0062] In the target sample, the type of the contamination site in the contamination source sample of the site in the third group is the heterozygous mutant site or the wild-type site;

[0063] In the target sample, the type of the contamination site in the fourth group of sites in the contamination source sample is the homozygous mutant site.

[0064] Preferably, obtaining the contamination source result corresponding to the sample to be screened according to the contamination site type and the mutation frequencies of the first group, the second group, the third group and the fourth group of the sample to be screened includes:

[0065] Determine whether the mutation frequency corresponding to each site in the first group, the second group, the third group, and the fourth group in the sample to be screened all conform to the contamination site type of the corresponding group in the target sample;

[0066] If yes, the contamination source result is determined as follows: the sample to be screened is the contamination source of the target sample;

[0067] If not, the contamination source result is determined as: the sample to be screened is not the contamination source of the target sample.

[0068] Preferably, the types of contamination sites that all conform to the corresponding groups in the target sample include:

[0069] All of the following conditions are met at the same time:

[0070] A. The mutation frequencies of the sites in the first group of samples to be screened are all within the range of 0%-1% of the wild-type sites;

[0071] B. The mutation frequency of the sites in the second group of samples to be screened is within the range of 30%-70% of the heterozygous mutant sites, or within the range of 99%-100% of the homozygous mutant sites;

[0072] C. The mutation frequency of the sites in the third group of samples to be screened is within the range of 30%-70% of the heterozygous mutant sites, or within the range of 0%-1% of the wild-type sites;

[0073] D. The mutation frequencies of the sites in the fourth group of samples to be screened are all within the range of 99%-100% of the homozygous mutant sites.

[0074] In addition, to solve the above problems, the present invention also provides a sequencing sample contamination analysis device, comprising:

[0075] A determination acquisition module is used to determine the SNP site of the target sample and obtain the mutation frequency corresponding to each SNP site of the target sample;

[0076] A classification and statistics module is used to classify the SNP sites of the target sample according to the mutation frequency, and obtain the site typing category corresponding to each of the SNP sites, and the statistical number corresponding to each of the site typing categories; wherein the site typing categories include wild-type sites, homozygous mutant sites and heterozygous mutant sites;

[0077] A number statistics module is used to determine the abnormal site category in the target sample and the statistical number corresponding to each abnormal site category based on the mutation frequency; wherein the abnormal site category includes WT contaminated sites and Hom contaminated sites;

[0078] The contamination analysis module is used to obtain the contamination analysis result of the SNP site according to the statistical number corresponding to the site typing category and the abnormal site category.

[0079] In addition, to solve the above problems, the present invention also provides a sequencing sample contamination analysis system, comprising a memory and a processor, wherein the memory stores a sequencing sample contamination analysis program, and the processor runs the sequencing sample contamination analysis program to enable the sequencing sample contamination analysis system to perform the sequencing sample contamination analysis method as described above.

[0080] In addition, to solve the above problems, the present invention also provides a computer-readable storage medium, on which a sequencing sample contamination analysis program is stored. When the sequencing sample contamination analysis program is executed by a processor, the sequencing sample contamination analysis method as described above is implemented.

[0081] The present invention provides a method, device, system and storage medium for analyzing contamination of sequencing samples, wherein the method comprises: determining the SNP sites of a target sample, and obtaining the mutation frequency corresponding to each SNP site of the target sample; classifying the SNP sites of the target sample according to the mutation frequency, obtaining the site typing category corresponding to each SNP site, and the statistical number corresponding to each site typing category; wherein the site typing categories include wild-type sites, homozygous mutant sites and heterozygous mutant sites; based on the mutation frequency, determining the abnormal site category in the target sample, and the statistical number corresponding to each abnormal site category; wherein the abnormal site category includes WT contaminated sites and Hom contaminated sites; and obtaining the contamination analysis result of the SNP site according to the site typing category and the statistical number corresponding to the abnormal site category. The present invention provides a method for analyzing contamination of sequencing samples. SNP sites are introduced into a gene detection panel, and suspected contaminated samples are judged by abnormal frequencies, so as to analyze whether the samples are contaminated and obtain contamination analysis results. For samples in a high-throughput sequencing process, on the one hand, an effective way is provided for analyzing and detecting sample contamination, and on the other hand, false positive results can be more effectively identified and excluded, thereby ensuring the accuracy of high-throughput sequencing data and the authenticity of experimental results. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 A schematic diagram of the structure of the hardware operating environment involved in the embodiment of the method for analyzing contamination of sequencing samples of the present invention;

[0083] Figure 2 This is a schematic diagram of the process of Example 1 of the method for analyzing sequencing sample contamination of the present invention;

[0084] Figure 3 Schematic diagram of VAF distribution of SNP sites in Example 1 of the method for analyzing contamination of sequencing samples of the present invention;

[0085] Figure 4 The box plot of the distribution of SNP sites in Example 1 of the method for analyzing contamination of sequencing samples of the present invention;

[0086] Figure 5 A schematic diagram of the mutation frequency region range involved in different sites in the site typing category and abnormal site category in Example 2 of the method for analyzing sequencing sample contamination of the present invention;

[0087] Figure 6 This is a schematic diagram of a detailed process of step S400 in Example 2 of the method for analyzing contamination of sequencing samples of the present invention;

[0088] Figure 7This is a supplementary flow chart of steps S450 and S460 (for determining the predicted contamination ratio) after step S440 in Example 3 of the method for analyzing contamination of sequencing samples of the present invention;

[0089] Figure 8 This is a supplementary flow chart of steps S470-S490 (for determining the number of contamination sources) after step S440 in Example 4 of the method for analyzing contamination of sequencing samples of the present invention;

[0090] Fig. 9 This is a schematic diagram of a detailed process of step S470 in Example 4 of the method for analyzing contamination of sequencing samples of the present invention;

[0091] Fig.10 This is a supplementary flow chart of steps S481-S484 (for determining the contamination source result) after step S480 in Example 5 of the method for analyzing contamination of sequencing samples of the present invention;

[0092] Fig.11 This is a schematic diagram of a detailed process of step S484 in Example 5 of the method for analyzing contamination of sequencing samples of the present invention;

[0093] Fig.12 Schematic diagram of module connection of a sequencing sample contamination analysis device in an embodiment of the present invention.

[0094] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0095] The embodiments of the present invention are described in detail below, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions.

[0096] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0097] In the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0098] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0099] like Figure 1 , which is a schematic diagram of the structure of the hardware operating environment of the terminal involved in the embodiment of the present invention.

[0100] The analysis system for sequencing sample contamination in the embodiment of the present invention may be a PC, or a mobile terminal device such as a smart phone, a tablet computer or a portable computer. The analysis system for sequencing sample contamination may include: a processor 1001, such as a CPU, a network interface 1004, a user interface 1003, a memory 1005 and a communication bus 1002. Among them, the communication bus 1002 is used to realize the connection and communication between these components. The user interface 1003 may include a display screen, an input unit such as a keyboard, and a remote control, and the optional user interface 1003 may also include a standard wired interface and a wireless interface. The network interface 1004 may optionally include a standard wired interface and a wireless interface (such as a WI-FI interface). The memory 1005 may be a high-speed RAM memory or a stable memory, such as a disk memory. The memory 1005 may also be a storage device independent of the aforementioned processor 1001. Optionally, the analysis system for sequencing sample contamination may also include an RF (Radio Frequency) circuit, an audio circuit, a WiFi module, and the like. In addition, the sequencing sample contamination analysis system may also be configured with other sensors such as a gyroscope, a barometer, a hygrometer, a thermometer, an infrared sensor, etc., which will not be described in detail here.

[0101] Those skilled in the art will understand that Figure 1 The sequencing sample contamination analysis system shown in the figure does not constitute a limitation thereof, and may include more or fewer components than shown in the figure, or combine certain components, or arrange the components differently. Figure 1 As shown, the memory 1005 as a computer-readable storage medium may include an operating system, a data interface control program, a network connection program, and a sequencing sample contamination analysis program.

[0102] In summary, the present application provides a method for analyzing contamination of sequencing samples. By introducing SNP sites into the gene detection panel and judging suspected contaminated samples by abnormal frequencies, the sample is analyzed to see whether it is contaminated and a contamination analysis result is obtained. For samples in the high-throughput sequencing process, on the one hand, it provides an effective way for the analysis and detection of sample contamination, and on the other hand, it can more effectively identify and exclude false positive results, thereby ensuring the accuracy of high-throughput sequencing data and the authenticity of experimental results.

[0103] Embodiment 1:

[0104] Reference Figure 2 , Example 1 of the present application provides a method for analyzing contamination of a sequencing sample, comprising:

[0105] Step S100, determining the SNP site of the target sample, and obtaining the mutation frequency corresponding to each SNP site of the target sample;

[0106] As mentioned above, SNP sites (Single Nucleotide Polymorphism) refer to the variation of a single nucleotide occurring at a specific position in the genome. This variation is usually manifested as differences between different individuals in the nucleotides (such as adenine (A), thymine (T), cytosine (C) or guanine (G)) at a certain position in the DNA sequence. SNP sites are the most common type of genetic variation in the genomes of humans and other organisms.

[0107] The target samples mentioned above are samples that need to be tested and analyzed.

[0108] As described above, a number of pre-confirmed SNP sites are added to the gene detection panel, and the number of SNP sites is preferably more than 100.

[0109] The purpose of the above steps is to identify and record all SNP sites in the target sample and determine the mutation frequency of each SNP site. Mutation frequency refers to the proportion of mutant alleles at the SNP position. High-throughput sequencing technology, such as next-generation sequencing (NGS), is used to determine the SNP sites in the sample and calculate the mutation frequency to obtain the specific mutation frequency of each SNP site, which is the basic data for subsequent analysis.

[0110] Through high-precision sequencing technology, mutations can be accurately identified, and even low-frequency mutations can be detected.

[0111] Specifically, modern bioinformatics software and tools, such as SAMtools, GATK, etc., can be used to assist in identifying SNP sites and calculating mutation frequencies.

[0112] As mentioned above, the SNP sites are determined, specifically, a number of SNP sites are added to the gene detection panel during the test. The number of SNP sites is preferably more than 100. In theory, the more the number, the more accurate and representative the statistics.

[0113] The SNP site selection condition is that the population frequency of the mutant allele is as close to 0.5 as possible (taking the 211 SNP sites screened by this laboratory as an example, the average population frequency of the mutant allele is 0.44). When the sample is not contaminated, the frequency distribution of the SNP site is concentrated in three areas: the three areas corresponding to the wild type, homozygous mutant type and heterozygous mutant type.

[0114] Step S200, classifying the SNP sites of the target sample according to the mutation frequency, obtaining a site typing category corresponding to each of the SNP sites, and a statistical number corresponding to each of the site typing categories; wherein the site typing categories include wild-type sites, homozygous mutant sites, and heterozygous mutant sites;

[0115] As described above, based on the mutation frequency (VAF) obtained in step S100, each SNP site is classified as wild type (mutation frequency close to 0%), homozygous mutant type (mutation frequency close to 100%) or heterozygous mutant type (mutation frequency between 30%-70%), as shown in the following table.

[0116] Table 1. Three locus typing categories and corresponding frequency distribution areas

[0117] NO. Locus typing category Abbreviation Frequency distribution area 1 wild type locus WT 0-1％ 2 Homozygous mutant loci Hom 30-70％ 3 Heterozygous mutant loci Het 99-100％

[0118] Each SNP locus is assigned to a specific typing category, and the number of loci in each typing category is counted. This classification helps to quickly identify possible contamination, because in non-contaminated samples, the typing categories should usually be highly consistent.

[0119] The classification process can be automatically performed by setting threshold conditions, and counting statistics can be performed for each category.

[0120] For example, in the present embodiment, 727 uncontaminated samples were counted, and the VAF distribution of these SNPs is as follows: Figure 3 shown.

[0121] The number of wild-type (WT) and homozygous mutant (Hom) SNP sites in these 727 samples was counted (statistical box diagram as shown in Figure 4 shown):

[0122] The mean number of WT sites was 73, with a standard deviation of 7;

[0123] The mean number of Hom sites was 48, with a standard deviation of 6;

[0124] The mean number of WT+Hom sites was 121, with a standard deviation of 9.

[0125] Step S300, based on the mutation frequency, determine the abnormal site category in the target sample and the statistical number corresponding to each abnormal site category; wherein the abnormal site category includes WT contaminated sites and Hom contaminated sites.

[0126] As described above, on the basis of known classifications, abnormal site categories are further identified, such as WT contamination sites (sites produced after wild type is contaminated) and Hom contamination sites (produced after homozygous sites are contaminated), and the deviation between the mutation frequency and the expected frequency is analyzed to determine whether it is contamination, and the category of abnormal sites and their number are obtained, which helps to identify the source and degree of contamination, thereby making the detection of sample contamination more accurate and specific.

[0127] Specifically, anomalies can be identified by comparing control samples or known data using statistical analysis methods.

[0128] Step S400, obtaining the contamination analysis result of the SNP site according to the statistical number corresponding to the site typing category and the abnormal site category.

[0129] The above analysis is combined to obtain the final contamination analysis results, indicating whether the sample is contaminated. The data and analysis of the previous steps are summarized to form a report. The detailed contamination analysis report, including the contamination site, type, possible source of contamination, etc., provides important quality control information for the further use of the sample.

[0130] Specifically, the data is integrated and analyzed and presented using visualization tools, such as the ggplot2 package in the R language.

[0131] For example, Bayesian statistical methods can be used to estimate the relationship between the mutation frequency of SNP sites and contamination, and combined with machine learning methods to classify and identify contamination. For example, a classifier can be trained to identify normal and contaminated SNP sites based on known contaminated and non-contaminated data in the training set.

[0132] In the above, the statistical numbers corresponding to the site typing categories and the abnormal site categories are used to obtain the results through comparison.

[0133] In summary, a method for analyzing sequencing sample contamination is provided in the embodiments of the present application. By introducing SNP sites into the gene detection panel and judging suspected contaminated samples by abnormal frequencies, whether the samples are contaminated is analyzed and a contamination analysis result is obtained. For samples in the high-throughput sequencing process, on the one hand, it provides an effective way for the analysis and detection of sample contamination, and on the other hand, it can more effectively identify and exclude false positive results, thereby ensuring the accuracy of high-throughput sequencing data and the authenticity of experimental results.

[0134] Embodiment 2:

[0135] Reference Figure 5 Embodiment 2 of the present application provides a method for analyzing contamination of sequencing samples, based on the above embodiment 1. The step S400, according to the statistical number corresponding to the site typing category and the abnormal site category, obtains the contamination analysis result of the SNP site, including:

[0136] Step S410, calculating the abnormality rate of the target sample according to the statistical number corresponding to the site typing category and the abnormal site category;

[0137] As described above, the abnormality rate of the target sample is calculated based on the statistical number of site typing categories and abnormal site categories.

[0138] Step S420, determining whether the abnormality rate of the target sample is not greater than 10%;

[0139] As mentioned above, it is necessary to determine whether the abnormality rate of the target sample is less than or equal to 10%.

[0140] Step S430, if the abnormality rate of the target sample is not greater than 10%, determining that the contamination analysis result is that the target sample is not contaminated;

[0141] Step S440: If the abnormality rate of the target sample is greater than 10%, it is determined that the contamination analysis result is that the target sample is contaminated.

[0142] If the abnormality rate is not greater than 10% in value, it is determined that the sample is not contaminated. If the abnormality rate is greater than 10% in value, it is determined that the sample is contaminated.

[0143] The above method provides a clear judgment standard to facilitate the rapid identification and processing of contaminated samples. Its specific implementation can be implemented through any programming environment that supports basic mathematical operations, such as Python or R, to calculate the abnormality rate and implement the judgment logic.

[0144] refer to Figure 6 , in the site typing category, the mutation frequency corresponding to the wild-type site is 0%-1%;

[0145] The mutation frequency corresponding to the homozygous mutant site is 30%-70%;

[0146] The mutation frequency corresponding to the heterozygous mutant site is 99%-100%;

[0147] In the abnormal site category, the mutation frequency corresponding to the WT contamination site is 1%-10%;

[0148] The mutation frequency corresponding to the Hom contaminated sites is 90%-99%.

[0149] As mentioned above, the mutation frequency of the wild-type site in the site typing category is 0%-1%, which means that there are almost no or very few mutations.

[0150] The mutation frequency of the homozygous mutant loci in the locus typing category is 99%-100%, which indicates that the same mutation occurs on both homologous chromosomes.

[0151] The mutation frequency of heterozygous mutant loci in the locus typing category is 30%-70%, indicating that the mutation only appears on one chromosome and the other is the wild-type allele.

[0152] In the abnormal site category, the mutation frequency of the WT contaminated site is 1%-10%. This situation indicates that there is a certain degree of contamination (the contamination ratio does not exceed 20%), because the mutation frequency is higher than the normal wild type, but does not reach the frequency of the heterozygous mutant type.

[0153] The mutation frequency of Hom contaminated sites in the abnormal site category is 90%-99%. This situation also indicates the presence of a certain degree of contamination (the contamination ratio does not exceed 20%), because the mutation frequency is lower than the normal homozygous mutant type, but does not reach the frequency of the heterozygous mutant type.

[0154] For sites with mutation frequencies of 10%-30% and 70-90%, the deviation in heterozygous mutation frequency may be caused by tumor polyploidy, the deviation may be caused by sequencing randomness, or a large proportion of contamination. Since their reliable sources cannot be determined, they are not considered in this method.

[0155] Specifically, data can be obtained using high-throughput sequencing technology, and frequencies can be calculated and classified using bioinformatics tools.

[0156] The result is an accurate classification of each SNP locus, which facilitates subsequent genetic analysis and disease diagnosis.

[0157] Furthermore, in step S410, the calculation method of the abnormality rate is:

[0158]

[0159] Among them, ratio represents the abnormal rate; YC1 represents the statistical number of the WT contaminated sites; YC2 represents the statistical number of the Hom contaminated sites; WT represents the statistical number of the wild-type sites; Hom represents the statistical number of the homozygous mutant sites.

[0160] As mentioned above, this formula (Formula 1) provides a quantitative method to estimate the degree of contamination in a sample by comparing the number of contaminated sites to the number of normal sites.

[0161] By implementing this formula, a percentage value can be obtained, which intuitively shows the degree of contamination of the sample, thereby providing a standardized method for assessing contamination. The implementation method can also use common data processing software or programming languages, such as the dplyr package of R language or the Numpy library of Python for calculation.

[0162] These steps as a whole constitute a systematic approach to assess and deal with contamination issues in sequencing samples, which not only improves data quality but also provides important support for genetic research and clinical applications.

[0163] Embodiment 3:

[0164] Reference Figure 7 Embodiment 3 of the present invention provides a method for analyzing contamination of sequencing samples. Based on the above embodiment 2, the step S440, after determining that the contamination analysis result is that the target sample has been contaminated, further includes:

[0165] Step S450, calculating the mean mutation frequency of the SNP sites with mutation frequencies in the range of 1%-10% in the target sample as a first mean; and calculating the mean mutation frequency of the SNP sites with mutation frequencies in the range of 90%-99% in the target sample as a second mean;

[0166] Step S460: determining a numerical range of the predicted contamination ratio corresponding to the target sample according to the first mean and the second mean.

[0167] After confirming that the sample was contaminated (i.e., the abnormality rate was greater than 10%), the means of the SNP sites within two specific mutation frequency ranges were further calculated: mutation frequencies between 1% and 10% (WT contaminated sites) and 90% and 99% (Hom contaminated sites).

[0168] The first mean is the average mutation frequency of SNP sites with mutation frequencies in the range of 1%-10%, and the second mean is the average mutation frequency of SNP sites with mutation frequencies in the range of 90%-99%.

[0169] As described above, the method provided in this embodiment is used to make a further prediction of the contamination ratio on the premise that it is determined that the target sample has been contaminated.

[0170] It should be noted that the method in this embodiment is aimed at predicting and analyzing the contamination ratio to be less than 20%, and the limitation is mainly due to the design of the analysis method and assumptions. These designs mainly consider relatively mild or primary stage contamination. The reason is that in this analysis, the mutation frequency intervals used to identify contamination are set at 1%-10% and 90%-99%. These thresholds are mainly intended to capture those situations where the mutation frequency deviates slightly from the wild type or homozygous mutant type due to contamination. If the mutation frequency is far away from these ranges, it is impossible to distinguish whether the mutation frequency is caused by contamination or is itself a heterozygous mutation.

[0171] When selecting the 1%-10% and 90%-99% mutation frequency intervals, this method assumes that the sites falling in these intervals correspond to 0% and 100% frequencies respectively when they are not contaminated. If there are a large number of sites in the 10%-30% and 70%-90% mutation frequency intervals, in addition to the possibility of a large proportion of contamination, it may also be caused by other biological reasons (such as tumor polyploidy).

[0172] Prevention and control in actual operation: In biological experiments and data analysis, data deviations within a small range (such as 1%-20% contamination) are more common and easier to handle. For contamination exceeding 20%, resequencing or more stringent sample processing and experimental design measures are usually considered, as this situation may seriously affect the reliability of the experimental results.

[0173] Therefore, this analysis method is mainly designed for those relatively mild contamination situations that can be estimated by the average mutation frequency. When dealing with situations with higher contamination ratios, more complex statistical models or molecular biology techniques may be needed to accurately evaluate and deal with these problems. If the contamination ratio is significantly greater than 20%, it may be necessary to consider more comprehensive data correction methods or re-acquire sample data.

[0174] Furthermore, determining the numerical range of the predicted contamination ratio corresponding to the target sample according to the first mean and the second mean includes:

[0175] The maximum and minimum extreme values ​​in the predicted pollution ratio are determined according to the following formula:

[0176]

[0177] C Het =C Hom ×2; (Formula 3)

[0178] The numerical range of the predicted contamination ratio of the target sample is: C Hom ≤C 预测 ≤C Het ;

[0179] Among them, C Hom The contamination ratio when all SNP sites representing the contamination source are homozygous mutant sites is taken as the minimum extreme value; C Het The contamination ratio when all SNP sites representing the contamination source are heterozygous mutant sites is taken as the maximum extreme value; represents the first mean; represents the second mean; C 预测 Represents the predicted contamination ratio of the target sample.

[0180] The above formulas (Formulas 2 and 3) calculate these two extreme values ​​to determine the possible range of pollution ratios, from minimum to maximum, thereby obtaining a range of predicted pollution ratios.

[0181] For example, for a target sample that is judged to be contaminated, the sites with mutation frequencies between 1-10% (the contaminated sample was originally WT, and the contamination source sample was Het / Hom) and 90-99% (the contaminated sample was originally Hom, and the contamination source sample was Het / WT) are counted, and the average mutation frequencies of the two groups of sites are calculated respectively. For example, if the target sample is sample21, the details are as follows:

[0182] (1) 1-10% group: there are 22 loci with frequencies of 1.6, 2.6, 1.2, 1.3, 1.8, 1.7, 3.2, 3.5, 1.1, 2.7, 3.8, 5.4, 2.2, 1.6, 2.7, 3.4, 1.4, 1.4, 1.8, 1.9, 2.7, and 1.2, respectively. The first mean is 2.28.

[0183] (2) 90-99% group: there are 30 loci with frequencies of 98.6, 97, 97.7, 98.6, 96.3, 98.4, 97.6, 96.8, 96.8, 96.6, 97.4, 95.1, 96.3, 96.7, 97, 95.9, 97, 98.5, 98.9, 96.7, 94.7, 97, 98.7, 98.5, 97.4, 96.1, 96.4, 96.9, 98.2, and 95.4, respectively. The calculated second mean is 97.1;

[0184] (3) Referring to the above pollution principle section, the pollution ratios under two extreme conditions are estimated:

[0185] If all the SNPs of the pollution source are homozygous (Hom), the pollution ratio is:

[0186]

[0187] (4) If all the SNPs of the pollution source are heterozygous (Het), the pollution ratio is twice as high as the former, that is: CHet =C Hom ×2=2.59%×2=5.18%;

[0188] In summary, the numerical range of the predicted contamination ratio corresponding to the target sample is between 2.59% and 5.18%.

[0189] Embodiment 4:

[0190] Reference Figure 8 Embodiment 4 of the present invention provides a method for analyzing contamination of sequencing samples. Based on the above embodiment 2, the step S440, after determining that the contamination analysis result is that the target sample has been contaminated, further includes:

[0191] Step S470, if the abnormality rate of the target sample is greater than 10%, a preset abnormality rate expected value-pollution source sample number correspondence relationship is obtained, and the abnormality rate of the target sample is compared with the abnormality rate expected value in the preset abnormality rate expected value-pollution source sample number correspondence relationship; wherein the preset abnormality rate expected value-pollution source sample number correspondence relationship includes the abnormality rate expected value and the predicted pollution source sample number corresponding to the abnormality rate expected value;

[0192] As mentioned above, if the abnormality rate of the target sample is less than or equal to 10%, it can be preliminarily inferred that the probability of contamination is small and can be ignored.

[0193] In this embodiment, the target samples whose abnormality rate is greater than 10% are mainly targeted. If the abnormality rate of the target sample is greater than 10%, a more in-depth analysis is performed.

[0194] The preset abnormality rate expected value-pollution source sample number correspondence relationship is a data correspondence relationship pre-constructed through a large amount of data, which includes the abnormality rate expected value and the predicted pollution source sample number corresponding to the abnormality rate expected value.

[0195] The relationship between the abnormality rate of a large number of data samples and the number of pollution source samples is used to construct the preset abnormality rate expected value-pollution source sample number correspondence, which is used as the evaluation standard to compare and screen the abnormality rate of the target samples, which can further improve the accuracy of data prediction.

[0196] Step S480: if the abnormality rate is equal to any one of the abnormality rate expected values ​​in the preset abnormality rate expected value-pollution source sample number correspondence relationship, the predicted pollution source sample number corresponding to the abnormality rate expected value is used as the pollution source number of the target sample;

[0197] Step S490, if the abnormality rate is not equal to any one of the WT-Hom site expected values ​​in the preset abnormality rate expected value-pollution source sample number correspondence, then the two abnormality rate expected values ​​before and after which are numerically close in the abnormality rate expected value are taken as the two end values, and the two predicted pollution source sample numbers corresponding to the two end values ​​are taken as the numerical range of the number of pollution sources of the target sample.

[0198] In the above, the possible number of pollution sources is estimated by comparing the abnormality rate of the target sample with the preset expected abnormality rate values ​​(these values ​​are associated with different numbers of pollution source samples). It is necessary to establish a relationship table between the expected abnormality rate and the number of pollution source samples in advance, which can be preset based on historical data or theoretical models, and compare the actual abnormality rate of the target sample with these preset values.

[0199] In the comparison process, it can be summarized as the following two situations:

[0200] (1) Case 1: If the abnormality rate of the target sample matches a value in the preset table, that is, is equal to it, the corresponding number of pollution sources can be directly obtained.

[0201] (2) Case 2: If there is no direct match, use the two preset values ​​at the closest ends to determine a range of possible pollution source quantities.

[0202] This method can provide a quantitative estimate of the number of pollution sources based on specific anomaly rate data, increase the flexibility and adaptability of the analysis, and adjust the preset values ​​according to different situations.

[0203] For example, the correspondence between the expected value of the preset abnormality rate and the number of pollution source samples is shown in the following table:

[0204] Table 2. Correspondence between the expected value of the preset abnormality rate and the number of pollution source samples

[0205] Number of pollution source samples 0 1 2 3 4 5 6 ∝ Expected value of abnormal rate 0％ 57.9％ 78.1％ 87％ 91.6％ 94.3 95.9％ 100％

[0206] Table 3. Results of the number of target samples and their corresponding predicted pollution source samples obtained through testing

[0207] Abnormality rate% A B C D Target sample 79.1 65.9 40 94.3 Predicted number of pollution source samples 2-3 1-2 0-1 5

[0208] By comparison, the predicted number of pollution source samples of target sample A is between 2-3; the predicted number of pollution source samples of target sample B is between 1-2; the abnormality rate of target sample C is greater than 10%, and the predicted number of pollution source samples is between 0-1, which is estimated to be 1; the abnormality rate of target sample D is 94.3%, which is equal to the expected value of the abnormality rate corresponding to 5 pollution source samples in the preset abnormality rate expected value-pollution source sample number correspondence relationship, so the predicted number of pollution source samples of target sample D is 5.

[0209] For further reference, Fig. 9 In step S470, the method for establishing the correspondence between the expected value of the abnormality rate and the number of pollution source samples includes:

[0210] Step S471, establish an uncontaminated sample group, determine the SNP sites that are the same as the target sample, and obtain the expected total number of wild-type sites and homozygous mutant sites of the uncontaminated samples in the uncontaminated sample group, as well as the population frequency of the wild-type allele and the population frequency of the mutant allele corresponding to the SNP site of each uncontaminated sample in the uncontaminated sample group.

[0211] In the above steps, the following data can be obtained:

[0212] (1) the expected total number of wild-type sites and homozygous mutant sites in the uncontaminated sample group;

[0213] (2) the population frequency of the wild-type allele corresponding to each SNP site of the uncontaminated sample in the uncontaminated sample group;

[0214] (3) The population frequency of the mutant allele corresponding to the SNP site of each uncontaminated sample in the uncontaminated sample group.

[0215] The calculation method of the expected value of the total number is:

[0216] Step (1) based on the SNP site, obtaining the number of WT+Hom sites of each uncontaminated sample;

[0217] Step (2) calculates the mean number of WT+Hom sites in the uncontaminated sample group as the expected total number.

[0218] Above, the population frequency of the wild-type allele (A) corresponding to the SNP site of each uncontaminated sample in the uncontaminated sample group; and the population frequency of the mutant allele (a) corresponding to the SNP site of each uncontaminated sample in the uncontaminated sample group, wherein the letters A and a represent two of the four bases.

[0219] As mentioned above, when establishing the correspondence between the expected value of the preset abnormality rate and the number of samples from the contamination source, it is necessary to examine and analyze the data of the uncontaminated samples. The confirmation condition of the SNP site is that the population frequency of the mutant allele is as close to 0.5 as possible (taking the 211 SNP sites screened in this embodiment as an example, the average population frequency of the mutant allele is 0.44).

[0220] In terms of the step logic of the method, an uncontaminated sample group can be first established, the SNP site can be determined, and then the preset abnormality rate expected value-contamination source sample number correspondence relationship can be obtained, and then step S100 can be performed. Alternatively, after determining that the contamination analysis result shows that the target sample has been contaminated, a preset abnormality rate expected value-contamination source sample number correspondence relationship can be established based on the determined SNP site, and then the target sample can be evaluated by obtaining the preset abnormality rate expected value-contamination source sample number correspondence relationship.

[0221] As mentioned above, in genetics and population genetics research, the population frequency of mutant alleles is a key statistical indicator, which reflects the relative frequency of a specific genotype or allele in a certain population or group. These data usually come from extensive genetics research and population genome projects, such as the 1000Genomes Project, HapMap Project, etc. These projects provide a lot of information about human genetic diversity through large-scale genome sequencing and analysis of different populations.

[0222] The population frequencies of wild-type and mutant alleles (represented by p and q, respectively, where the population frequency of wild-type alleles is p and the population frequency of mutant alleles is q) are the basis for genetic analysis and research. They reflect the prevalence of a genotype in a specific population and are key parameters for calculating genetic risk, disease association, and other genetic properties.

[0223] Knowing these frequencies is important when performing genetic pollution analysis or SNP analysis because they can help predict changes in genotype frequencies under specific circumstances, such as when pollution occurs. SNP sites with mutant allele population frequencies as close to 0.5 are selected to ensure that the sites have high polymorphism, which allows small changes in frequency to be detected more sensitively in statistical analysis. When p is close to 0.5, genetic variation is more evenly distributed in the population, which helps improve the sensitivity and accuracy of the analysis.

[0224] In genetic research or clinical diagnosis, the rational selection of representative SNP loci is key, which requires making decisions based on detailed population frequency data. These data can be obtained not only from the databases and studies mentioned above, but also through the laboratory's own research and collection, especially when it comes to specific ethnic groups or populations.

[0225] In summary, the population frequencies of the wild-type allele and the mutant allele, i.e., p and q, are selected based on extensive population genetics data and are aimed at ensuring efficient and sensitive genetic analysis.

[0226] Step S472, calculating the comprehensive probability of abnormal frequency after each SNP site is contaminated by pollution sources with different predicted pollution source sample numbers according to the population frequency of the wild-type allele and the population frequency of the mutant allele;

[0227] The population frequencies of wild-type and mutant SNP sites mentioned above can be obtained from public databases such as dbSNP, 1000Genomes Project, etc., or obtained through previous laboratory research.

[0228] After obtaining the population frequencies of wild-type and mutant SNP sites, the comprehensive probability of abnormal frequencies that each SNP site may exhibit under the influence of different numbers of pollution source samples is calculated. The Hardy-Weinberg equilibrium formula in genetics can be used to calculate the probability of abnormal frequencies of each site under different pollution conditions.

[0229] It should be noted that the Hardy-Weinberg equilibrium (HWE) is a basic concept in population genetics. This equilibrium state describes an idealized, undisturbed population in which the genotype frequency remains constant between generations. The Hardy-Weinberg equilibrium can be described by the following mathematical expression:

[0230] Let p and q represent the frequencies of the two alleles at a certain locus, where p+q=1. According to the Hardy-Weinberg equilibrium, the frequency of the genotype at the locus can be calculated according to the following formula:

[0231] The frequency of wild-type pp is p 2 ;

[0232] The frequency of heterozygous mutant pq is 2pq;

[0233] The frequency of the homozygous mutant qq is q 2 .

[0234] Step S473, for each number of samples of the predicted pollution source, the comprehensive probability of all SNPs producing abnormal frequencies is summed up to obtain the expected value of the number of abnormal sites produced by the uncontaminated sample after it is assumed to be contaminated;

[0235] As mentioned above, by summing up the abnormal frequency probabilities in various situations, we can obtain the possible impact of a pollution source sample population. By summing up all the calculated abnormal frequency probabilities, we can get the expected value of the number of abnormal sites.

[0236] Step S474, calculating the expected value of the abnormality rate corresponding to the uncontaminated sample according to the expected value of the number of abnormal sites and the expected value of the total number of wild-type sites and homozygous mutant sites in the uncontaminated sample;

[0237] Step S475, constructing a corresponding relationship between the preset abnormality rate expected value and the number of pollution source samples according to different predicted pollution source sample numbers and the expected value of the abnormality rate corresponding to the predicted pollution source sample number.

[0238] According to the expected value of the number of abnormal sites and the expected value of the total number of wild-type and homozygous sites in uncontaminated samples, the expected abnormality rate is calculated, and the correspondence between the preset expected value of the abnormality rate and the number of samples from the contamination source is constructed. In this step, an expected value is provided for the predicted abnormality rate in each contamination case, which can be used to evaluate the contamination status of the actual sample.

[0239] Furthermore, the step S472 calculates the comprehensive probability of abnormal frequency of each SNP site after being contaminated by pollution sources with different predicted pollution source sample numbers based on the population frequency of the wild-type allele and the population frequency of the mutant allele, including:

[0240] (1) When the number of samples of the predicted pollution source is 1, the comprehensive probability of abnormal frequency after being polluted by the pollution source is calculated according to the following formula:

[0241] Prob1=2×p×q×(1-q); (Formula 4)

[0242] (2) When the number of samples of the predicted pollution source is greater than 1, the comprehensive probability of abnormal frequency after being polluted by the pollution source is calculated according to the following formula:

[0243] Prob n =1-(p 2(n+1) +2pq+q 2(n+1) ); (Formula 5)

[0244] Where n represents the number of samples of the predicted pollution source; Prob n It represents the comprehensive probability of abnormal frequency after being contaminated by the pollution source when the number of samples of the pollution source is predicted to be n; p represents the population frequency of the wild-type allele; q represents the population frequency of the mutant allele.

[0245] The above part involves calculating the combined probability of abnormal frequency for each SNP locus under the influence of different numbers of contamination source samples. This calculation is based on the Hardy-Weinberg law in genetics and assumes that genetic alleles are randomly distributed in the population.

[0246] A mathematical model was established to estimate the probability of SNP sites showing abnormal frequencies under different pollution conditions. The Hardy-Weinberg law was used to calculate the probability of a specific SNP site showing abnormal frequencies under different pollution conditions (different numbers of pollution source samples).

[0247] Furthermore, in step S473, the comprehensive probability of abnormal frequency corresponding to each number of predicted pollution source samples is summed up to obtain the expected value of the number of abnormal sites generated by the uncontaminated sample after it is assumed to be contaminated, including:

[0248] The expected value of the number of abnormal sites is calculated by the following expression:

[0249]

[0250] Among them, S 总 represents the expected value of the number of abnormal sites; i represents the number of the SNP site; Prob i It represents the comprehensive probability of abnormal frequency after being contaminated by the pollution source when the SNP site is i; N represents the total number of SNP sites.

[0251] For each of the predicted pollution source sample numbers, the sum of the comprehensive probabilities of abnormal frequency is:

[0252] S 总 =Prob1+Prob2+Prob3……Prob N ;

[0253] Thus, the expected value of the number of abnormal sites is obtained.

[0254] Furthermore, the step S474, calculating the expected value of the abnormal rate corresponding to the uncontaminated sample according to the expected value of the number of abnormal sites and the total number of wild-type sites and homozygous mutant sites in the uncontaminated sample, includes:

[0255] The expected value of the abnormal rate is calculated by the following formula:

[0256]

[0257] Among them, ratio 期望 represents the expected value of the abnormal rate of the uncontaminated sample after it is assumed to be contaminated; S 总 represents the expected value of the number of abnormal sites; N WT+Hom Represents the expected total number of wild-type sites and homozygous mutant sites in the uncontaminated sample.

[0258] For example, the population frequency of wild-type A of a certain SNP is p = 0.6, and the population frequency of mutant T is q = 0.4;

[0259] The proportion of AA type (WT) in the population is 0.36 (the frequency of AA type (WT): p 2 =0.6 2=0.36), the proportion of AT type (Het) in the population is 0.48 (the frequency of AT type (Het): 2pq = 2 × 0.6 × 0.4 = 0.48) and the proportion of TT type (Hom) in the population is 0.16 (q 2 =0.4 2 =0.16).

[0260] The probability that the contaminated sample is WT and the contaminated sample is Het is 0.36×0.48=0.1728. The four possible combinations of abnormal frequencies and their corresponding occurrence probabilities are shown in the following table:

[0261] Table 4. Four possible combinations of abnormal frequencies

[0262]

[0263] That is, for a site with a mutant population frequency of 0.4, the comprehensive probability of producing an abnormal frequency after contamination is 0.1728+0.0576+0.0576+0.0768=0.3648. Extending to the general case, the comprehensive probability of a site with a mutant population frequency of p producing an abnormal frequency after contamination is:

[0264] When n=1, the calculation process is as follows according to Formula 4:

[0265] Prob1=2×p×q×(1-p×q)=2×0.6×0.4×(1-0.6×0.4)=0.3648.

[0266] When n=3, according to Formula 5, the calculation process is as follows:

[0267] Prob3 = 1-(p 2(3+1) +2pq+q 2(3+1) )=1-(p 8 +2pq+q 8 )=1-(0.6 8 +2×

[0268] 0.6×0.4+0.48=0.5025.

[0269] Furthermore, the combined probability of all SNPs producing abnormal frequencies is summed up to obtain the expected value of the number of abnormal sites produced by the uncontaminated sample after it is assumed to be contaminated. Based on the data in this example, the following calculation is performed:

[0270] When the number of pollution source samples is 1:

[0271] S 总 =0.255+0.311+0.372……Prob 211 =70.071;

[0272] Furthermore, the expected value of the abnormal rate of the uncontaminated sample after being assumed to be contaminated is calculated based on the expected value of the number of abnormal sites and the total number of wild-type sites and homozygous mutant sites in the uncontaminated sample:

[0273] The total number of wild-type sites and homozygous mutant sites in the uncontaminated sample is 121;

[0274]

[0275] Similarly, the expected value of the anomaly rate when the number of pollution source samples is greater than 1 can be calculated in turn to obtain the corresponding relationship between the expected value of the anomaly rate and the number of pollution source samples in Table 2 above.

[0276] Embodiment 5:

[0277] Reference Fig.10 Embodiment 5 of the present invention provides a method for analyzing sequencing sample contamination. Based on the above embodiment 4, in step S480, if the abnormality rate is equal to any one of the abnormality rate expected values ​​in the preset abnormality rate expected value-contamination source sample number correspondence relationship, then the predicted number of contamination source samples corresponding to the abnormality rate expected value is used as the number of contamination sources of the target sample, and further includes:

[0278] Step S481, if the number of the pollution sources is equal to 1, obtaining the sample to be screened and the mutation frequency corresponding to each of the SNP sites in the sample to be screened;

[0279] As mentioned above, when it is determined that a sample is affected by a single source of contamination, how to determine the specific source of contamination. The above method further analyzes and confirms the source of contamination by comparing the SNP site mutation frequency of the target sample with the possible source of contamination sample.

[0280] The samples to be screened are possible contamination source samples, which will be compared with the target samples to determine the contamination source. A series of potential contamination source samples are collected, and the mutation frequency data of the same SNP sites in these samples are obtained.

[0281] Step S482, grouping the mutation frequencies of the SNP sites of the target sample and the sample to be screened to obtain a first group, a second group, a third group and a fourth group respectively; wherein the mutation frequency of the first group ranges from 0% to 1%; the mutation frequency of the second group ranges from 1% to 10%; the mutation frequency of the third group ranges from 90% to 99%; the mutation frequency of the fourth group ranges from 99% to 100%;

[0282] As described above, the SNP mutation frequencies in the target sample and the sample to be screened are classified according to different frequency ranges for ease of analysis and comparison.

[0283] Specifically, the grouping and mutation frequencies are:

[0284] (1) Group 1: 0%-1%;

[0285] (2) Group 2: 1%-10%;

[0286] (3) Group 3: 90%-99%;

[0287] (4) Group 4: 99%-100%.

[0288] Step S483, determining the types of contamination sites corresponding to the first group, the second group, the third group and the fourth group in the target sample; the types of contamination sites include wild-type sites, homozygous mutant sites and heterozygous mutant sites;

[0289] As mentioned above, in the target samples, the contamination site types corresponding to the first group, the second group, the third group and the fourth group are the corresponding relationships between the groups and the contamination site types.

[0290] Thus, the type of loci in each group (wild type, homozygous mutation, heterozygous mutation) is identified, which helps to understand the nature of the contamination source. Specifically, based on the frequency distribution of SNP loci, the possible type of loci in each group (such as homozygous, heterozygous) is determined.

[0291] Step S484, obtaining the contamination source results corresponding to the samples to be screened according to the contamination site type and the mutation frequencies of the first group, the second group, the third group and the fourth group of the samples to be screened.

[0292] In the above, it is determined whether the sample to be screened is the actual source of contamination. By comparing the frequencies and types of each group of sites in the target sample and the sample to be screened, it is determined whether all frequencies and types match.

[0293] Through careful grouping and comparison, the pollution source can be accurately identified, and clear grouping and comparison criteria make the implementation of this method simple and accurate.

[0294] Furthermore, in the target samples, the types of contamination sites corresponding to the first group, the second group, the third group and the fourth group include:

[0295] (1) In the target sample, the type of the contamination site in the contamination source sample of the site in the first group is the wild-type site;

[0296] (2) In the target sample, the type of the contamination site in the contamination source sample at the site in the second group is the heterozygous mutant site or the homozygous mutant site;

[0297] (3) In the target sample, the type of the contamination site in the contamination source sample is the heterozygous mutant site or the wild-type site;

[0298] (4) In the target sample, the type of the contamination site in the fourth group of sites in the contamination source sample is the homozygous mutant site.

[0299] Table 5. Groups and corresponding contamination site types

[0300]

[0301]

[0302] The above provides a correspondence between grouping and contamination site type. Among them, the first group (0%-1%): homozygous mutant sites. The second group (1%-10%): heterozygous or homozygous mutant sites. The third group (90%-99%): heterozygous or homozygous mutant sites. The fourth group (99%-100%): homozygous mutant sites.

[0303] Check whether the sites of each mutation frequency group in the pre-selected sample match the site types of the corresponding group in the target sample. Compare the types of sites in each mutation frequency group in the target sample and the pre-selected sample to confirm whether they all match. In this way, it is determined whether the pre-selected sample (sample to be screened) is the source of contamination of the target sample. If the site types in all groups match the types of the target sample, the pre-selected sample is considered to be the source of contamination.

[0304] For further reference, Fig.11 The step S484, according to the contamination site type and the mutation frequencies of the first group, the second group, the third group and the fourth group of the sample to be screened, obtains the contamination source result corresponding to the sample to be screened, including:

[0305] Step S4841, determining whether the mutation frequencies corresponding to each site in the first group, the second group, the third group, and the fourth group in the sample to be screened all conform to the contamination site type of the corresponding group in the target sample;

[0306] As mentioned above, the mutation frequencies of the SNP sites of the target sample are divided into four groups: 0%-1%, 1%-10%, 90%-99%, and 99%-100%. Each group corresponds to a predefined contamination site type (such as homozygous, heterozygous), and then compared with the corresponding sites in the sample to be screened to determine whether the SNP sites in the sample to be screened show contamination characteristics consistent with the contamination source that contaminates the target sample.

[0307] Step S4842: If yes, the result of determining the source of contamination is: the sample to be screened is the source of contamination of the target sample;

[0308] Step S4843, if not, then the result of determining the source of contamination is: the sample to be screened is not the source of contamination of the target sample.

[0309] If all SNP mutation frequency groups of the sample to be screened completely match the type of contamination sites in the target sample, the sample to be screened is considered to be a source of contamination.

[0310] If any non-compliance occurs, the sample to be screened is considered not to be the source of contamination.

[0311] In the above steps, the contamination source is determined by comparing the SNP site mutation frequency of the sample to be screened with that of the target sample, which has the following advantages:

[0312] Accuracy and specificity: This method can accurately identify which samples are potential sources of contamination by directly comparing the mutation frequency of SNP sites with predefined contamination site types (homozygous, heterozygous, etc.). This precise match provides highly specific results and reduces the possibility of false positives.

[0313] Rapid Identification: The method allows for rapid identification and determination of the source of contamination, which helps to take timely measures to treat or re-process the sample, thereby ensuring the accuracy and reliability of the experimental data.

[0314] Easy to operate: By grouping and comparing SNP mutation frequencies, this method is easy to operate and can be easily implemented through existing bioinformatics tools and statistical software without the need for complex experimental operations.

[0315] Scalability and adaptability: This technical solution is not limited to specific types of SNP analysis, but can also be extended to the analysis of other genetic markers. In addition, the definition and classification criteria of contamination site types can be adjusted according to the needs of different studies to make it more adaptable.

[0316] Cost savings: By identifying the source of contamination in a timely manner, experimental repetitions due to data contamination can be avoided, thus saving a lot of re-experimentation and material costs.

[0317] Enhance data confidence: Accurately identifying and excluding contaminated samples can improve the confidence of experimental data and the reliability of research results, which is particularly important in fields such as high-throughput sequencing and precision medicine.

[0318] Furthermore, in step S4841, all the contamination site types that meet the corresponding grouping in the target sample include:

[0319] All of the following conditions are met at the same time:

[0320] A. The mutation frequencies of the sites in the first group of samples to be screened are all within the range of 0%-1% of the wild-type sites;

[0321] B. The mutation frequency of the sites in the second group of samples to be screened is within the range of 30%-70% of the heterozygous mutant sites, or within the range of 99%-100% of the homozygous mutant sites;

[0322] C. The mutation frequency of the sites in the third group of samples to be screened is within the range of 30%-70% of the heterozygous mutant sites, or within the range of 0%-1% of the wild-type sites;

[0323] D. The mutation frequencies of the sites in the fourth group of samples to be screened are all within the range of 99%-100% of the homozygous mutant sites.

[0324] Table 5. Groups and corresponding contamination site types and value ranges

[0325]

[0326] For example, the target sample is simple21, the sample to be analyzed is sample2, and the data is as follows:

[0327] (1) Based on condition A, for the first group:

[0328] Table 6. Mutation frequencies of the first group (0%-1%) of 29 sites in the target sample and the corresponding sites in the samples to be analyzed

[0329]

[0330] It can be seen from the above data that the mutation frequencies of the sites in the first group of samples to be screened (sample 2) are all within the range of 0%-1% of the wild-type sites.

[0331] It should be noted that the data in the same row in the table represent the mutation frequencies of two samples sample2 and sample21 at the same site, the same below.

[0332] (2) Based on condition B, for the second group:

[0333] Table 7. Mutation frequencies of the second group (1%-10%) of 22 sites in the target samples and the corresponding sites in the samples to be analyzed

[0334] NO. sample2 sample21 NO. sample2 sample21 1 39.8 1.6 12 99.8 5.4 2 49.6 2.6 13 37.6 2.2 3 40.9 1.2 14 36.7 1.6 4 43.5 1.3 15 100 2.7 5 49.1 1.8 16 61.2 3.4 6 46.8 1.7 17 54.2 1.4 7 48.4 3.2 18 61.6 1.4 8 100 3.5 19 99.7 1.8 9 36.9 1.1 20 100 1.9 10 100 2.7 21 50.6 2.7 11 99.9 3.8 22 57 1.2

[0335] From the above data, it can be seen that the mutation frequency of the sites in the first group of samples to be screened (sample 2) is within the range of 30%-70% of the heterozygous mutant sites, or within the range of 99%-100% of the homozygous mutant sites;

[0336] (3) Based on condition C, for the third group:

[0337] Table 8. Mutation frequencies of the third group (90%-99%) of 30 sites in the target samples and the corresponding sites in the samples to be analyzed

[0338] NO. sample2 sample21 NO. sample2 sample21 1 53.2 98.6 16 39.1 95.9 2 0.1 97 17 0.2 97 3 38 97.7 18 58.3 98.5 4 58.4 98.6 19 20.6 98.9 5 0 96.3 20 0 96.7 6 48.6 98.4 21 0 94.7 7 40.5 97.6 22 0 97 8 35.7 96.8 23 60.8 98.7 9 0 96.8 24 33.1 98.5 10 42 96.6 25 38.8 97.4 11 40.3 97.4 26 0 96.1 12 0 95.1 27 0 96.4 13 0 96.3 28 45.9 96.9 14 0 96.7 29 59.5 98.2 15 0.3 97 30 0 95.4

[0339] From the above data, it can be seen that the mutation frequency of the sites in the first group of samples to be screened (sample 2) is within the range of 30%-70% of the heterozygous mutant sites, or within the range of 0%-1% of the wild-type sites;

[0340] (4) Based on condition D, for the fourth group:

[0341] Table 9. Mutation frequencies of the 16 loci in the fourth group (99%-100%) of target samples and the corresponding loci in the samples to be analyzed

[0342]

[0343]

[0344] It can be seen from the above data that the mutation frequencies of the sites in the first group of samples to be screened (sample 2) are all within the range of 99%-100% of the homozygous mutant sites.

[0345] From the judgment results of the above four conditions A, B, C, and D, it can be seen that the mutation frequencies corresponding to each site in the first group, the second group, the third group, and the fourth group in the sample to be screened (sample2) all conform to the contamination site type of the corresponding group in the target sample (sample21). Therefore, the result of judging the source of contamination is: the sample to be screened is the source of contamination of the target sample.

[0346] In addition, reference Fig.12 This embodiment also provides a sequencing sample contamination analysis device, including:

[0347] A determination acquisition module 10 is used to determine the SNP site of the target sample and obtain the mutation frequency corresponding to each SNP site of the target sample;

[0348] A classification and statistics module 20 is used to classify the SNP sites of the target sample according to the mutation frequency, and obtain the site typing category corresponding to each of the SNP sites, and the statistical number corresponding to each of the site typing categories; wherein the site typing categories include wild-type sites, homozygous mutant sites and heterozygous mutant sites;

[0349] A number statistics module 30 is used to determine the abnormal site category in the target sample and the statistical number corresponding to each abnormal site category based on the mutation frequency; wherein the abnormal site category includes WT contaminated sites and Hom contaminated sites;

[0350] The contamination analysis module 40 is used to obtain the contamination analysis result of the SNP site according to the statistical number corresponding to the site typing category and the abnormal site category.

[0351] In addition, this embodiment also provides a sequencing sample contamination analysis system, including a memory and a processor, the memory stores a sequencing sample contamination analysis program, and the processor runs the sequencing sample contamination analysis program to enable the sequencing sample contamination analysis system to perform the sequencing sample contamination analysis method described above.

[0352] In addition, this embodiment also provides a computer-readable storage medium, on which a sequencing sample contamination analysis program is stored. When the sequencing sample contamination analysis program is executed by a processor, the sequencing sample contamination analysis method as described above is implemented.

[0353] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0354] Through the description of the above implementation methods, those skilled in the art can clearly understand that the above-mentioned embodiment methods can be implemented by means of software plus a necessary general hardware platform, and of course by hardware, but in many cases the former is a better implementation method. Based on such an understanding, the technical solution of the present invention is essentially or the part that contributes to the prior art can be embodied in the form of a software product, and the computer software product is stored in a storage medium (such as ROM / RAM, disk, CD) as described above, including a number of instructions for a terminal device (which can be a mobile phone, computer, server, or network device, etc.) to execute the methods described in each embodiment of the present invention. The above is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the present invention specification and drawings, or directly or indirectly used in other related technical fields, are similarly included in the patent protection scope of the present invention.

Claims

1. A method for analyzing contamination of sequencing samples, characterized in that: include: Determine the SNP site of the target sample, and obtain the mutation frequency corresponding to each SNP site of the target sample; The SNP sites of the target sample are classified according to the mutation frequency to obtain the site typing category corresponding to each SNP site and the statistical number corresponding to each site typing category; wherein the site typing category includes wild-type sites, homozygous mutant sites and heterozygous mutant sites; in the site typing category, the mutation frequency corresponding to the wild-type site is 0%-1%; the mutation frequency corresponding to the heterozygous mutant site is 30%-70%; the mutation frequency corresponding to the homozygous mutant site is 99%-100%; Based on the mutation frequency, determine the abnormal site category in the target sample and the statistical number corresponding to each abnormal site category; wherein the abnormal site category includes WT contamination site and Hom contamination site; the WT contamination site and the Hom contamination site are different from the range of the wild-type site, the homozygous mutant site and the heterozygous mutant site; and, in the abnormal site category, the mutation frequency VAF corresponding to the WT contamination site WT 1% ≤ VAF WT <10%; the mutation frequency VAF corresponding to the Hom contaminated site Hom 90%<VAF Hom ≤99%; According to the statistical number corresponding to the site typing category and the abnormal site category, the abnormal rate of the target sample is calculated, and the contamination analysis result of the SNP site is obtained based on the abnormal rate; wherein the calculation method of the abnormal rate is: Among them, ratio represents the abnormal rate; YC1 represents the statistical number of the WT contaminated sites; YC2 represents the statistical number of the Hom contaminated sites; WT represents the statistical number of the wild-type sites; Hom represents the statistical number of the homozygous mutant sites.

2. The method for analyzing sequencing sample contamination according to claim 1, characterized in that: The contamination analysis result of the SNP site obtained based on the abnormal rate includes: Determining whether the abnormality rate of the target sample is no greater than 10%; If the abnormality rate of the target sample is not greater than 10%, the contamination analysis result is determined to be that the target sample is not contaminated; If the abnormality rate of the target sample is greater than 10%, the contamination analysis result is determined to be that the target sample is contaminated.

3. The method for analyzing sequencing sample contamination according to claim 2, characterized in that: After determining that the contamination analysis result is that the target sample has been contaminated, the method further includes: Calculating the mean mutation frequency of the SNP sites with a mutation frequency in the range of 1%-10% in the target sample as a first mean; and calculating the mean mutation frequency of the SNP sites with a mutation frequency in the range of 90%-99% in the target sample as a second mean; Determine a numerical range of the predicted contamination ratio corresponding to the target sample according to the first mean and the second mean; The determining, according to the first mean and the second mean, a numerical range of the predicted contamination ratio corresponding to the target sample includes: The maximum and minimum extreme values ​​in the predicted pollution ratio are determined according to the following formula: C Het =C Hom ×2; The numerical range of the predicted contamination ratio of the target sample is: C Hom ≤C 预测 ≤C Het ; Among them, C Hom The contamination ratio when all SNP sites representing the contamination source are homozygous mutant sites is taken as the minimum extreme value; C Het The contamination ratio when all SNP sites representing the contamination source are heterozygous mutant sites is taken as the maximum extreme value; represents the first mean; represents the second mean; C 预测 Represents the predicted contamination ratio of the target sample.

4. The method for analyzing sequencing sample contamination according to claim 2, characterized in that: After determining that the contamination analysis result is that the target sample has been contaminated, the method further includes: If the abnormality rate of the target sample is greater than 10%, a preset abnormality rate expected value-pollution source sample number correspondence relationship is obtained, and the abnormality rate of the target sample is compared with the abnormality rate expected value in the preset abnormality rate expected value-pollution source sample number correspondence relationship; wherein the preset abnormality rate expected value-pollution source sample number correspondence relationship includes the abnormality rate expected value and the predicted pollution source sample number corresponding to the abnormality rate expected value; If the abnormality rate is equal to any one of the abnormality rate expected values ​​in the preset abnormality rate expected value-pollution source sample number correspondence relationship, the predicted pollution source sample number corresponding to the abnormality rate expected value is used as the pollution source number of the target sample; If the abnormality rate is not equal to any of the WT-Hom site expected values ​​in the preset abnormality rate expected value-contamination source sample number correspondence, then the two abnormality rate expected values ​​before and after which are numerically similar in the abnormality rate expected value are taken as the two end values, and the two predicted contamination source sample numbers corresponding to the two end values ​​are taken as the numerical range of the number of contamination sources of the target sample.

5. The method for analyzing sequencing sample contamination according to claim 4, characterized in that: The method for establishing the correspondence between the preset abnormality rate expected value and the number of pollution source samples includes: Establish an uncontaminated sample group, determine the SNP sites that are the same as the target sample, and obtain the expected total number of wild-type sites and homozygous mutant sites of the uncontaminated samples in the uncontaminated sample group, as well as the population frequency of the wild-type allele and the population frequency of the mutant allele corresponding to the SNP site of each uncontaminated sample in the uncontaminated sample group; wherein the calculation method of the total expected number is: based on the SNP site, obtain the number of WT+Hom sites of each uncontaminated sample; calculate the mean number of the WT+Hom sites in the uncontaminated sample group as the total expected number; According to the population frequency of the wild-type allele and the population frequency of the mutant allele, the comprehensive probability of each SNP site producing an abnormal frequency after being contaminated by contamination sources with different predicted numbers of contamination source samples is calculated; For each number of samples of the predicted pollution source, the comprehensive probability of all SNPs producing abnormal frequencies is summed up to obtain the expected value of the number of abnormal sites produced by the uncontaminated sample after it is assumed to be contaminated; The expected value of the abnormality rate of the uncontaminated sample after being assumed to be contaminated is calculated according to the expected value of the number of abnormal sites and the expected value of the total number of wild-type sites and homozygous mutant sites in the uncontaminated sample; According to different predicted numbers of pollution source samples and the expected value of the abnormality rate corresponding to the predicted number of pollution source samples, a corresponding relationship between the preset expected value of abnormality rate and the number of pollution source samples is constructed.

6. The method for analyzing sequencing sample contamination according to claim 5, characterized in that: The comprehensive probability of abnormal frequency of each SNP site after being contaminated by pollution sources with different predicted pollution source sample numbers is calculated based on the population frequency of the wild-type allele and the population frequency of the mutant allele, including: When the number of samples of the predicted pollution source is 1, the comprehensive probability of abnormal frequency after being polluted by the pollution source is calculated according to the following formula: Prob1 = 2 × p × q × (1-p × q); When the number of samples of the predicted pollution source is greater than 1, the comprehensive probability of abnormal frequency after being polluted by the pollution source is calculated according to the following formula: Prob n =1-(p 2(n+1) +2pq+q 2(n+1) ); Where n represents the number of samples of the predicted pollution source; Prob n It represents the comprehensive probability of abnormal frequency after being contaminated by the pollution source when the number of samples of the pollution source is predicted to be n; p represents the population frequency of the wild-type allele; q represents the population frequency of the mutant allele.

7. The method for analyzing sequencing sample contamination according to claim 5, characterized in that: The summation of the comprehensive probabilities of the abnormal frequencies corresponding to each number of samples of the predicted pollution source, to obtain the expected value of the number of abnormal sites generated by the uncontaminated sample after the sample is assumed to be contaminated, includes: The expected value of the number of abnormal sites is calculated by the following expression: Among them, S 总 represents the expected value of the number of abnormal sites; i represents the number of the SNP site; Prob i It represents the comprehensive probability of abnormal frequency after being contaminated by the pollution source when the SNP site is i; N represents the total number of SNP sites.

8. The method for analyzing sequencing sample contamination according to claim 5, characterized in that: The method of calculating the expected value of the abnormal rate corresponding to the uncontaminated sample according to the expected value of the number of abnormal sites and the total number of wild-type sites and homozygous mutant sites in the uncontaminated sample comprises: The expected value of the abnormal rate is calculated by the following formula: Among them, ratio 期望 represents the expected value of the abnormal rate of the uncontaminated sample after it is assumed to be contaminated; S 总 represents the expected value of the number of abnormal sites; N WT+Hom Represents the expected total number of wild-type sites and homozygous mutant sites in the uncontaminated sample.

9. The method for analyzing sequencing sample contamination according to claim 4, characterized in that: If the abnormality rate is equal to any one of the abnormality rate expected values ​​in the preset abnormality rate expected value-pollution source sample number correspondence relationship, then after taking the predicted pollution source sample number corresponding to the abnormality rate expected value as the pollution source number of the target sample, the method further includes: If the number of the pollution sources is equal to 1, then obtaining the sample to be screened and the mutation frequency corresponding to each of the SNP sites in the sample to be screened; The mutation frequencies of the SNP sites of the target sample and the sample to be screened are grouped to obtain a first group, a second group, a third group and a fourth group, respectively; wherein the mutation frequency of the first group ranges from 0% to 1%; the mutation frequency of the second group ranges from 1% to 10%; the mutation frequency of the third group ranges from 90% to 99%; and the mutation frequency of the fourth group ranges from 99% to 100%; Determine the types of contamination sites corresponding to the first group, the second group, the third group, and the fourth group in the target sample; the types of contamination sites include wild-type sites, homozygous mutant sites, and heterozygous mutant sites; Obtaining a contamination source result corresponding to the sample to be screened according to the contamination site type and the mutation frequencies of the first group, the second group, the third group, and the fourth group of the sample to be screened; In the target samples, the types of contamination sites corresponding to the first group, the second group, the third group and the fourth group include: In the target sample, the type of the contamination site in the contamination source sample of the site in the first group is the wild-type site; In the target sample, the type of the contamination site in the contamination source sample of the site in the second group is the heterozygous mutant site or the homozygous mutant site; In the target sample, the type of the contamination site in the contamination source sample of the site in the third group is the heterozygous mutant site or the wild-type site; In the target sample, the type of the contamination site in the fourth group of sites in the contamination source sample is the homozygous mutant site; The step of obtaining the contamination source result corresponding to the sample to be screened according to the contamination site type and the mutation frequencies of the first group, the second group, the third group and the fourth group of the sample to be screened comprises: Determine whether the mutation frequency corresponding to each site in the first group, the second group, the third group, and the fourth group in the sample to be screened all conform to the contamination site type of the corresponding group in the target sample; If yes, the contamination source result is determined as follows: the sample to be screened is the contamination source of the target sample; If not, the contamination source result is determined as follows: the sample to be screened is not the contamination source of the target sample; The types of contamination sites that all meet the corresponding grouping in the target sample include: All of the following conditions are met at the same time: A. The mutation frequencies of the sites in the first group of samples to be screened are all within the range of 0%-1% of the wild-type sites; B. The mutation frequency of the sites in the second group of samples to be screened is within the range of 30%-70% of the heterozygous mutant sites, or within the range of 99%-100% of the homozygous mutant sites; C. The mutation frequency of the sites in the third group of samples to be screened is within the range of 30%-70% of the heterozygous mutant sites, or within the range of 0%-1% of the wild-type sites; D. The mutation frequencies of the sites in the fourth group of samples to be screened are all within the range of 99%-100% of the homozygous mutant sites.

10. A sequencing sample contamination analysis device, characterized in that: include: A determination acquisition module is used to determine the SNP site of the target sample and obtain the mutation frequency corresponding to each SNP site of the target sample; A classification and statistics module is used to classify the SNP sites of the target sample according to the mutation frequency, and obtain the site typing category corresponding to each of the SNP sites, and the statistical number corresponding to each of the site typing categories; wherein the site typing categories include wild-type sites, homozygous mutant sites and heterozygous mutant sites; in the site typing categories, the mutation frequency corresponding to the wild-type site is 0%-1%; the mutation frequency corresponding to the heterozygous mutant site is 30%-70%; the mutation frequency corresponding to the homozygous mutant site is 99%-100%; A number statistics module is used to determine the abnormal site category in the target sample and the statistical number corresponding to each abnormal site category based on the mutation frequency; wherein the abnormal site category includes WT contamination site and Hom contamination site; in the abnormal site category, the mutation frequency VAF corresponding to the WT contamination site WT 1% ≤ VAF WT <10%; the mutation frequency VAF corresponding to the Hom contaminated site Hom 90%<VAF Hom ≤99%; The contamination analysis module is used to calculate the abnormality rate of the target sample according to the statistical number of the site typing category and the abnormal site category, and obtain the contamination analysis result of the SNP site based on the abnormality rate; wherein the calculation method of the abnormality rate is: Among them, ratio represents the abnormal rate; YC1 represents the statistical number of the WT contaminated sites; YC2 represents the statistical number of the Hom contaminated sites; WT represents the statistical number of the wild-type sites; Hom represents the statistical number of the homozygous mutant sites.

11. A sequencing sample contamination analysis system, characterized in that: It comprises a memory and a processor, wherein the memory stores a sequencing sample contamination analysis program, and the processor runs the sequencing sample contamination analysis program to enable the sequencing sample contamination analysis system to perform the sequencing sample contamination analysis method as described in any one of claims 1 to 9.

12. A computer-readable storage medium, characterized in that: The computer-readable storage medium stores a sequencing sample contamination analysis program, and when the sequencing sample contamination analysis program is executed by the processor, the sequencing sample contamination analysis method according to any one of claims 1 to 9 is implemented.

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