WO2018214010A1 - Method, device, and storage medium for detecting mutation on the basis of sequencing data - Google Patents

Abstract

A method, a device, and a storage medium for detecting mutation on the basis of sequencing data. The method comprises: aligning sequencing data of a plurality of individuals from the same population to a reference genome and conducting mutation detection, to obtain alignment position and mutation information in read lengths; for mutated sites, respectively summing the total mutated site depths and mutated base-type depths of all of the individuals in the population, and then calculating sub-allelic frequencies and/or Hardy-Weinberg equilibriums of the mutated sites in the population; plotting a diagram of the relationship between the total mutated site depths and/or the mutated base-type depths of the mutated sites and dbSNP%, and filtering the total mutated site depths and/or the mutated base-type depths as well as the sub-allelic frequencies and/or the Hardy-Weinberg equilibriums of the mutated sites; and filtering the mutation information of each individual, to obtain final mutation information of each individual. The present application can use low-depth sequencing data of populations for accurate mutation detection, improving data utilization.

Description

Method, device and storage medium for detecting variation based on sequencing data Technical field

The present invention relates to the field of bioinformatics technology, and in particular, to a mutation detection method, apparatus and storage medium based on sequencing data.

Background technique

Although the cost of sequencing is still declining, there is still no qualitative breakthrough in the sequencing strategy, and the cost of high-depth sequencing data remains high. Therefore, based on the low depth variation detection method, the utilization rate of the existing low-depth whole genome sequencing data will be effectively improved. At present, the detection of low-depth mutations is still carried out using conventional software, and the obtained variation information is often filtered out in the quality control due to insufficient depth.

In order to obtain the variation information of these low-depth regions, Genotype imputation based on gene-linked inference is now mainly used, and high-depth variation sites are used as marker sites, and existing gene collections are referenced (a certain population is collected). Gene information), inferring unknown mutation information based on the linkage relationship between known marker sites and adjacent unknown sites. This method uses the genetic linkage relationship to infer, and the inferred method fills the obtained unknown sites. In fact, it also covers in the individual sequencing process, but it is neglected due to the low depth, resulting in waste of these data. The genotyping is done by inference method, and the real base situation obtained by sequencing is not considered. The genetic information in the simple reference gene set cannot obtain the unique variation of the individual itself, and the accuracy and low depth data are sacrificed. Moreover, since genotypic inference filling techniques need to use known high-depth variation site information, there is a requirement for variation information density. If the number of known mutation sites is too small and the distribution density is too low, it will be difficult to carry out the gene. Type inferred to fill. In addition, the genotype filling technique takes a long time and has a poor timeliness.

Summary of the invention

The invention provides a mutation detection method, device and storage medium based on sequencing data, which can directly perform accurate mutation detection by using low-depth sequencing data of a group, and improve data utilization.

According to a first aspect, an embodiment provides a variation detection method based on sequencing data, including:

Sequencing data of a plurality of individuals derived from the same population are compared to a reference genome and subjected to mutation detection to obtain a read length alignment position and variation information;

According to the above alignment position and mutation information, the total depth of the variation sites and the depth of the variant bases of each individual in the population are respectively added to each variation site, and then the sub-alleles of each variation site in the population are calculated. Frequency and / or Hardy Weinberg balance, get the cumulative results of group variation information;

Mapping the total depth of the variant site and/or the depth of the variant base type to the dbSNP%, and based on the set filter threshold, the total depth of the variant site and/or the depth of the variant base type and the above allele frequency and / or Hardy Weinberg balances the filtering to filter out the value below the above filtering threshold, and obtains the accumulated result of the filtered population variation information;

The variation information of each individual is filtered according to the accumulated result of the filtered population variation information, and the final variation information of each individual is obtained.

According to a second aspect, an embodiment provides a variation detection apparatus based on sequencing data, including:

The comparison and mutation detecting device is configured to compare the sequencing data of the plurality of individuals from the same group to the reference genome and perform mutation detection to obtain the read length alignment position and the variation information;

Adding and calculating means for summing the total depth of the variant sites and the depth of the variant bases of all individuals in the population according to the above-mentioned alignment position and variation information, and then calculating the respective variation sites The sub-allelic frequency in the population and/or the Hardy Weinberg equilibrium, the cumulative result of the population variation information is obtained;

A population variation information filtering device for plotting the total depth of the variant site and/or the depth of the variant base type and the dbSNP%, and based on the set filtering threshold, the total depth of the variant site and/or the depth of the variant base And the above allele frequencies and/or Hardy Weinberg equilibrium are filtered to filter out below Filtering the value of the threshold to obtain the accumulated result of the filtered population variation information;

The individual variation information filtering device is configured to filter the variation information of each individual according to the accumulated result of the filtered population variation information to obtain final variation information of each individual.

According to a third aspect, an embodiment provides a variation detection apparatus based on sequencing data, including:

Memory for storing programs;

a processor for implementing the following method by executing the program stored in the above memory:

Sequencing data of a plurality of individuals derived from the same population are compared to a reference genome and subjected to mutation detection to obtain a read length alignment position and variation information;

According to the above alignment position and mutation information, the total depth of the variation sites and the depth of the variant bases of each individual in the population are respectively added to each variation site, and then the sub-alleles of each variation site in the population are calculated. Frequency and / or Hardy Weinberg balance, get the cumulative results of group variation information;

Mapping the total depth of the variant site and/or the depth of the variant base type to the dbSNP%, and based on the set filter threshold, the total depth of the variant site and/or the depth of the variant base type and the above allele frequency and / or Hardy Weinberg balances the filtering to filter out the value below the above filtering threshold, and obtains the accumulated result of the filtered population variation information;

The variation information of each individual is filtered according to the accumulated result of the filtered population variation information, and the final variation information of each individual is obtained.

According to a fourth aspect, an embodiment provides a computer readable storage medium comprising a program executable by a processor to implement the following method:

Sequencing data of a plurality of individuals derived from the same population are compared to a reference genome and subjected to mutation detection to obtain a read length alignment position and variation information;

According to the above alignment position and mutation information, the total depth of the variation sites and the depth of the variant bases of each individual in the population are respectively added for each variation site, and then each variation site is calculated in the population. The sub-allelic frequency and/or Hardy Weinberg equilibrium, resulting in cumulative results of population variation information;

Mapping the total depth of the variant site and/or the depth of the variant base type to the dbSNP%, and based on the set filter threshold, the total depth of the variant site and/or the depth of the variant base type and the above allele frequency and / or Hardy Weinberg balances the filtering to filter out the value below the above filtering threshold, and obtains the accumulated result of the filtered population variation information;

The variation information of each individual is filtered according to the accumulated result of the filtered population variation information, and the final variation information of each individual is obtained.

The invention can utilize the low-depth sequencing data of the population, and directly performs accurate mutation detection without using chain inference to improve data utilization, and the variation information obtained in the low-depth region can be used as a marker site to help improve the existing linkage inference. result.

DRAWINGS

1 is a flow chart of a method for detecting variation based on sequencing data according to an embodiment of the present invention;

2 is a structural block diagram of a variation detecting apparatus based on sequencing data according to an embodiment of the present invention;

3 is a graph showing the relationship between the depth of the variant base type and the dbSNP% in one embodiment of the present invention, wherein each curve represents a different chromosome, the bold curve represents the average value, and the gray portion represents the standard deviation.

detailed description

The present invention will be further described in detail below with reference to the accompanying drawings. In the following embodiments, many of the details are described in order to provide a better understanding of the invention. However, those skilled in the art can easily realize that some of the features may be omitted in different situations, or may be replaced by other components, materials, and methods. In some instances, some of the operations related to the present invention have not been shown or described in the specification in order to avoid that the core portion of the present invention is overwhelmed by excessive description, and those skilled in the art will describe these in detail. Related operations are not necessary They can fully understand the relevant operations according to the description in the manual and the general technical knowledge in the field.

In addition, the features, operations, or characteristics described in the specification may be combined in any suitable manner to form various embodiments. At the same time, the steps or actions in the method description can also be sequentially changed or adjusted in a manner that can be apparent to those skilled in the art. Therefore, the various sequences in the specification and the drawings are only for the purpose of describing a particular embodiment, and are not intended to

For the detection of mutations in low-depth sequencing data, the traditional method is directed to a single sample. The method of the present invention combines the low-depth variations of the population together as a variation information of a single sample, thereby realizing the conversion of low-depth groups into fictitious highs. Depth individuals, and then carry out mutation detection, obtain accurate mutation detection results and then split to obtain the variation information of individual individuals.

As shown in FIG. 1, a method for detecting a variation based on sequencing data according to an embodiment of the present invention includes the following steps:

Step S101: Aligning the sequencing data of the plurality of individuals from the same population to the reference genome and performing mutation detection to obtain the read position and the variation information of the Reads.

In the embodiment of the present invention, the sequencing data belongs to the same group, for example, the same species (human, pig, etc.). These sequencing data may be offline data of the same batch, or may be offline data belonging to the same group although belonging to different batches. The format of the sequencing data is, for example, the Fastq format. The reference genome can be published genomic data for each population (species), for example, for a human, the reference genome can be the human reference genome hg19. The comparison software can be a commonly used BWA software, etc., and the mutation detection software can be a commonly used GATK software. The obtained variation information includes elements such as the chromosome, position, and variant base type in which the mutation is located. Regarding the number of individuals (ie, the sample size), the embodiments of the present invention are not strictly required, but the more the sample size, the more favorable to find more mutation sites.

In the embodiment of the present invention, the average sequencing depth of the sequencing data is not particularly limited, and the method of the embodiment of the present invention is particularly suitable for low-depth sequencing data, such as sequencing data having an average depth of 1× to 15×. In In one embodiment of the invention, the average sequencing depth of the sequencing data is 3.5x.

Step S102: According to the obtained alignment position and variation information, sum the total depth of the variation sites and the depth of the variant bases of each individual in the population for each variation site, and then calculate the variation sites in the population. The Minor allele frequency (MAF) and/or the Hardy-Weinberg equilibrium (HWE), the cumulative result of the population variation information is obtained, and the so-called "group variation information accumulation result" includes each variation site. The total depth of the additive site, the depth of the variant base, and the MAF and/or HWE.

As used herein, the term "total depth of variation site" refers to the number of sequencing reads (Reads) of all individuals covering the site of variation; the term "mutation base type depth" refers to the The number of sequencing reads of all individuals of a particular base type at the variant site. It can be seen that for a particular variant site, the "total depth of the variant site" is the sum of the "mutation base depths" of the various base types. For example, if a variant site has two base types, A and T, and 100 of the sequencing reads in all individuals, 100 are A at the mutation site, and 100 are T at the mutation site. Then, the "mutation base type depth" of the two base types A and T is 100, and the "variation site total depth" of the mutation site is 200.

As used herein, the term "secondary allele frequency" refers to the frequency of unusual alleles in a given population; the term "Hardy Weinberg equilibrium" refers to the frequency of each allele The genotype frequencies of the alleles are stable in the inheritance, that is, the gene balance is maintained.

The method of the embodiments of the present invention is particularly suitable for single nucleotide polymorphism (SNP) variation, insertion/deletion (Ins/Del) mutation detection, and thus in a preferred embodiment of the invention, all individuals in the population When the total depth of the variant site and the depth of the variant base are respectively added, the three bases and other multibase mutations are removed and only the single nucleotide polymorphism variation is retained.

Step S103: plot the total depth of the variant site and/or the depth of the variant base type and the dbSNP%, and according to the set filtering threshold, the total depth of the variant site and/or the depth of the variant base type and the frequency of the minor allele. And/or Hardy Weinberg balances the filtering to filter out the value below the filtering threshold, and obtains the accumulated result of the filtered population variation information.

As used herein, the term "dbSNP%" refers to the proportion of found variant sites in the oligonucleotide polymorphism database and is commonly used to measure the accuracy of detected SNPs.

In an embodiment of the invention, the filtering threshold includes a filtering threshold of the total depth of the mutation site and/or a filtering threshold of the variant base type depth, and a filtering threshold of the MAF and/or a filtering threshold of the HWE. That is, the key indicators are the mutated base type depth (or total variation site depth), the MAF value, and/or the HWE value. The depth of the variant base type of a base type is directly related to the credibility of the mutation. HWE and MAF are often used to remove low-reliability variant sites.

In one embodiment of the invention, the filter threshold for the depth of the variant base type is 30x and the filter threshold for the MAF is 0.05. That is to say, the depth of the variant base type is greater than or equal to 30×, and the MAF is greater than 0.05, which is considered to be statistically significant.

In other embodiments of the present invention, the depth of filtration (variation base type depth or total variation site depth) may be appropriately adjusted according to the actual depth density distribution. Wherein, the "actual depth density distribution" may refer to the distribution of the sequencing depth of different mutation sites.

In other embodiments of the present invention, the sample size may be set according to the average depth of the data, for example, the average depth is 1×, and the sample size should be 30, so that the variation site type depth can reach a set value of 30× or more. Filter conditions. The more sample size, the more favorable it is to detect more variant sites.

Step S104: Filter the variation information of each individual according to the accumulated result of the filtered population variation information, and obtain the final variation information of each individual.

In a specific embodiment of the present invention, filtering the variation information of each individual specifically includes: if the existing chromosomes, locations, and variant base types in the original variation information of the individual are consistent with the cumulative result of the filtered population variation information. As a result, the result is retained, otherwise the result is filtered out. In addition, if there are more than two variations in one variant site, the variant site is filtered out.

Through the above steps, the final variation information of each individual can be obtained. The accuracy of the variation can then be assessed by dbSNP% and subsequent inferential filling and research analysis.

The method of the present invention improves data utilization in low-depth regions of high-throughput sequencing data, which would otherwise Neglected data is converted into usable data, and the accuracy of detecting SNPs is significantly improved, and the method is simple and easy to use, which can improve the accuracy and increase the data utilization rate, and can also be added to the previous step of traditional variation filling. Improve traditional methods.

Those skilled in the art can understand that all or part of the functions of the various methods in the above embodiments may be implemented by hardware or by a computer program. When all or part of the functions in the above embodiments are implemented by a computer program, the program may be stored in a computer readable storage medium, and the storage medium may include: a read only memory, a random access memory, a magnetic disk, an optical disk, a hard disk, etc. The computer executes the program to implement the above functions. For example, the program is stored in the memory of the device, and when the program in the memory is executed by the processor, all or part of the above functions can be realized. In addition, when all or part of the functions in the above embodiment are implemented by a computer program, the program may also be stored in a storage medium such as a server, another computer, a magnetic disk, an optical disk, a flash disk or a mobile hard disk, and may be saved by downloading or copying. The system is updated in the memory of the local device, or the system of the local device is updated. When the program in the memory is executed by the processor, all or part of the functions in the above embodiments may be implemented.

Therefore, an embodiment of the present invention further provides a mutation detecting device based on sequencing data, as shown in FIG. 2, including:

The comparison and variation detecting device 201 is configured to compare the sequencing data of the plurality of individuals from the same group to the reference genome and perform mutation detection to obtain the read length matching position and the variation information; the sum and calculation device 202, According to the alignment position and variation information, the total depth of the variation sites and the depth of the variant bases of each individual in the population are respectively added for each variation site, and then the sub-equations of each variation site in the population are calculated. The gene frequency and/or Hardy Weinberg equilibrium, the cumulative result of the population variation information is obtained; the population variation information filtering device 203 is used to map the total depth of the variant site and/or the relationship between the depth of the variant base and the dbSNP%, and The set filter threshold is filtered to filter the total depth of the variant site and/or the depth of the variant base type and the minor allele frequency and/or Hardy Weinberg equilibrium to filter out values below the filtration threshold. The group variation information accumulation result; the individual variation information filtering device 204 is configured to filter the variation information of each individual according to the accumulated result of the filtered group variation information, Each The final variation of the individual.

Another embodiment of the present invention further provides a variation detecting apparatus based on sequencing data, including:

Memory for storing programs;

a processor for implementing the following method by executing the program stored in the above memory:

The sequencing data of multiple individuals from the same population are compared to the reference genome and the mutation is detected to obtain the read position and the variation information of the read length; according to the alignment position and the variation information, the individual mutation sites are The total depth of the variant sites and the depth of the variant bases are summed separately, and then the sub-allelic frequencies and/or Hardy Weinberg equilibrium of each variant site in the population are calculated, and the cumulative result of the population variation information is obtained; Mapping the total depth of the variant site and/or the depth of the variant base type to the dbSNP% and based on the set filter threshold for the total depth of the variant site and/or the depth of the variant base and the frequency of the minor allele and/or Hardy Weinberg balances the filtering to filter out the value below the filtering threshold, and obtains the accumulated result of the filtered population variation information; according to the accumulated result of the filtered population variation information, the variation information of each individual is filtered to obtain each The final variation of the individual.

Yet another embodiment of the present invention also provides a computer readable storage medium comprising a program executable by a processor to implement the following method:

The sequencing data of multiple individuals from the same population are compared to the reference genome and the mutation is detected to obtain the read position and the variation information of the read length; according to the alignment position and the variation information, the individual mutation sites are The total depth of the variant sites and the depth of the variant bases are summed separately, and then the sub-allelic frequencies and/or Hardy Weinberg equilibrium of each variant site in the population are calculated, and the cumulative result of the population variation information is obtained; Mapping the total depth of the variant site and/or the depth of the variant base type to the dbSNP% and based on the set filter threshold for the total depth of the variant site and/or the depth of the variant base and the frequency of the minor allele and/or Hardy Weinberg balances the filtering to filter out the value below the filtering threshold, and obtains the accumulated result of the filtered population variation information; according to the accumulated result of the filtered population variation information, the variation information of each individual is filtered to obtain each The final variation of the individual.

The technical solutions and effects of the present invention are described in detail below through embodiments, and it should be understood that the embodiments are only It is intended to be illustrative, and not to limit the scope of the invention.

Example

In this example, the sample is sequencing data captured in 105 human MHC regions (4.9M region on chromosome 6), and the data concentrated in the MHC region accounts for about 50% of the data. That is to say, about 50% of the data is accompanied by other locations in the genome. Relatively speaking, this part of the data is low-depth data. Using the method of the present invention to detect the variation of the 50% low-depth region, the accuracy of the results is evaluated, and the 105 samples are also subjected to exon sequencing. In the exon sequencing data, the exon region is High depth. By comparing the consistency of low depth data and exon sequencing in the exon region, the consistency of the variation can be determined to assess the accuracy of the method of the invention.

The specific steps of this embodiment are as follows:

(1) Download the human reference genome hg19, use the BWA software to compare the sequencing read length to the reference genome hg19, and use GATK software to detect the mutation information, and lower the threshold to retain more variation information.

(2) For the sequencing data variation site captured by the MHC region, this embodiment studies with a low depth region, and the average depth of the low depth region in this embodiment is 3.5×. According to the chromosome, position and variant base type of the mutation site, the total depth of the mutation sites of 105 MHC samples and the depth of the variant base were respectively added to remove the three bases and other multibase mutations and calculate each The MAF value of the mutation site in the population obtained the cumulative result of the population variation information of 105 samples.

(3) Calculate the total depth of the variant site, the depth of the variant base, and the MAF value (some results are shown in Table 1), and calculate the data distribution form of the relevant values. It was found that the depth of dbSNP% and the variant base type reached 30×, and after analyzing the variation site and the MAF value distribution, the threshold of the total depth of the mutation site was set to 30×, the threshold of MAF was 0.05, and the value below the threshold was filtered out. , the filtered population variation information is added to the cumulative result. Fig. 3 is a graph showing the relationship between the depth of the mutant base type and the dbSNP% in the present embodiment.

Table 1

(4) According to the cumulative result of the population variation information filtered by 105 samples, the variation result of each individual is filtered. If there is a result that the chromosome, position, and variant base type are consistent with the filtered population variation information accumulation result in the original variation information of the individual, the result is retained, otherwise the result is filtered out. In addition, if there are more than two variations in one variant site, the variant site is filtered out.

(5) dbSNP% evaluates the accuracy of the variation, and found that dbSNP% can reach 90.3% (if the Y chromosome is removed and the coverage of chromosome 9 is dbSNP% is 93%). In addition, compared with the corresponding high-depth exon sequencing data, the consistency can reach 96.45% after comparison. The results are shown in Table 2. The number in the table indicates the number of 105 samples, and the table indicates “unprocessed”. Consistent data for comparison of the results obtained using the method of the invention with corresponding high depth exon sequencing data, the expression "this method" in the table indicates the results obtained using the method of the invention and the corresponding high depth exon sequencing data Contrast consistent data.

Table 2

The invention has been described above with reference to specific examples, which are merely intended to aid the understanding of the invention and are not intended to limit the invention. For the person skilled in the art to which the invention pertains, several simple derivations, variations or substitutions can be made in accordance with the inventive concept.

Claims (10)

A variation detection method based on sequencing data, characterized in that it comprises: Sequencing data of a plurality of individuals derived from the same population are compared to a reference genome and subjected to mutation detection to obtain a read length alignment position and variation information; According to the alignment position and the variation information, the total depth of the variation sites and the depth of the variant bases of each individual in the population are respectively added for each variation site, and then the sub-equations of each variation site in the population are calculated. Gene frequency and / or Hardy Weinberg equilibrium, get the cumulative results of group variation information; Mapping the total depth of the variant site and/or the depth of the variant base type to the dbSNP%, and based on the set filter threshold, the total depth of the variant site and/or the depth of the variant base and the sub-allele The frequency and/or Hardy Weinberg balance is filtered to filter out values below the filtration threshold to obtain a cumulative result of the filtered population variation information; The mutation information of each individual is filtered according to the accumulated result of the filtered population variation information, and the final variation information of each individual is obtained. The variation detecting method according to claim 1, wherein the average sequencing depth of the sequencing data is 1 × to 15 ×. The method of detecting variation according to claim 1, further comprising: removing three bases and other multibases when summing the total depth of the variant sites and the depth of the variant bases of all individuals in the population, respectively. Mutants retain only single nucleotide polymorphism variants. The mutation detecting method according to claim 1, wherein the filtering threshold includes a filtering threshold of the total depth of the mutation site and/or a filtering threshold of the variant base type depth, and the sub-equivalent The filtering threshold of the gene frequency and the filtering threshold of the Hardy Weinberg equilibrium. The variation detecting method according to claim 4, wherein the filtering threshold of the variation base type depth is 30×, and the filtering threshold of the secondary allele frequency is 0.05, the Hardy Weinberg equilibrium The filtering threshold is 0.01. The variation detecting method according to claim 1, wherein said each individual The filtering of the variation information specifically includes: If there is a result that the chromosome, position, and variant base type are consistent with the filtered population variation information accumulation result in the original variation information of the individual, the result is retained, otherwise the result is filtered out. The variation detection method according to claim 6, wherein the filtering the variation information of each individual further comprises: If there are more than two variants at one of the variant sites, the variant site is filtered out. A variation detecting device based on sequencing data, comprising: The comparison and mutation detecting device is configured to compare the sequencing data of the plurality of individuals from the same group to the reference genome and perform mutation detection to obtain the read length alignment position and the variation information; Adding and calculating means for summing the total depth of the variant sites and the depth of the variant bases of all individuals in the population according to the alignment position and the variation information, and then calculating the respective mutation positions The sub-allelic frequency and/or Hardy Weinberg equilibrium in the population is obtained, and the cumulative result of the population variation information is obtained; A population variation information filtering device for plotting the total depth of the variant site and/or the relationship between the depth of the variant base and the dbSNP%, and the total depth and/or variant base type of the variant site according to the set filtering threshold Depth and the sub-allelic frequency and/or Hardy Weinberg equilibrium are filtered to filter out values below the filtration threshold to obtain a cumulative result of the filtered population variation information; The individual variation information filtering device is configured to filter the variation information of each individual according to the accumulated result of the filtered population variation information to obtain final variation information of each individual. A variation detecting device based on sequencing data, comprising: Memory for storing programs; A processor for performing the method of the memory storage to implement the method of any one of claims 1 to 7. A computer readable storage medium, comprising a program executable by a processor to implement the method of any one of claims 1 to 7.

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