CN117672363A - Method, device and storage medium for detecting point mutation parent source - Google Patents
Method, device and storage medium for detecting point mutation parent source Download PDFInfo
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Abstract
The application discloses a method, a device and a storage medium for detecting a point mutation parent source. The method comprises the steps of obtaining a comparison result, carrying out genotype analysis, and judging parent sources according to genotype and genetic rules; if the parent is unable to judge, judging the parent source of the mutation site by analyzing whether the family has a base definitely derived from one of the mutated chain or the non-mutated chain; for the new mutation situation, whether the parent source of the mutation site is definitely judged by analyzing whether the chain with mutation or the chain without mutation of the precursor exists on the base definitely derived from one party, if not, the adjacent family heterozygous mutation site is searched, the parent source of the adjacent heterozygous site is judged by utilizing the method that both parents are heterozygous mutation situations, and further the parent source of the new mutation is deduced. The method can detect the parent sources of complex mutation and new mutation, and the detection result is accurate and reliable.
Description
Technical Field
The application relates to the technical field of point mutation parent source detection, in particular to a method, a device and a storage medium for detecting point mutation parent sources.
Background
At present, the second generation sequencing is the most widely used sequencing scheme, and has the advantages of high throughput and rapid sequencing, and can adapt to the requirement of mass gene sequencing in the current scientific research or medical industry. Analysis of second generation sequencing data often provides a rich information for scientific research or disease diagnosis, and mutation detection is a very important step in analysis of sequencing results, which can provide information of all point mutations occurring in a sequencing sample. Some point mutations occurring in the prover genome that are highly relevant to pathogenesis are the primary concern in current genetic testing procedures.
In addition, the relevance of the disease to the point mutation can be further confirmed according to whether the mutation is new or inherited from parents. However, in the conventional analysis procedure of the point mutation, the genetic source of the point mutation, i.e., the parent source, is not deeply analyzed, and thus some important information for causing the disease may be omitted.
The human genome is two copies of autosomes except for sex chromosomes, and under normal genetic conditions, the autosomes of the prover are inherited one from each parent, and if there is a mutation on the chromosome inherited from a parent, the prover will acquire the mutation at that position. In most cases, the mutation of the prover is inherited from one or both parents, and the rate of occurrence of new mutation of the prover is low. There are many factors that may cause a new mutation, but the new mutation is often considered as a serious pathogenic mutation in genetic disease analysis.
For some simple cases, such as where a mutation is present in only one party of the parent, it may be directly judged that the mutation originated from the party with the mutation. However, in the actual judgment, many cases are complicated, for example, the first-evidence person and the parent are both heterozygous mutations, and the first-evidence person has new mutations; there is currently no method for parental source detection for these complications.
Thus, how to detect more accurately and effectively the parental source of point mutations remains a problem to be solved in the art.
Disclosure of Invention
It is an object of the present application to provide a method, apparatus and storage medium of novel parent sources of point mutations at detection.
In order to achieve the above purpose, the present application adopts the following technical scheme:
a first aspect of the present application discloses a method of detecting a point mutation parent source comprising the steps of:
the comparison result obtaining step comprises the step of obtaining second-generation sequencing comparison results of ancestral three persons of a forerunner and a parent containing the position of the mutation site to be detected, namely, the result formed by comparing the original second-generation sequencing results to the matching position on the reference genome;
genotype analysis, namely, analyzing the genotype of mutation at a mutation site to be detected, namely, homozygosity or heterozygosity, and the genotypes of parents at the site according to a second-generation sequencing comparison result;
The preliminary parent source judging step comprises the steps of carrying out preliminary parent source judging according to genotypes and the following rules: (1) The mutation site to be detected is homozygous mutation, and the parent sources are judged to be both parents; (2) The mutation site to be detected is heterozygous mutation, one of parents of the site has no mutation, and the other parent has mutation, so that the mutation site to be detected is derived from the mutant parent; (3) The mutation site to be detected is heterozygous mutation, one of parents of the site is homozygous mutation, the other parent is heterozygous mutation, and the mutation site to be detected is derived from one of homozygous mutation; (4) The mutation site to be detected is heterozygous mutation, and both parents at the site are heterozygous mutation, and the parent heterozygous mutation is marked as a parent heterozygous site; (5) Marking the mutation site to be detected as new mutation if both parents have no mutation at the corresponding site of the mutation site;
the parent heterozygous site source judging step comprises the steps of respectively extracting all comparison results containing mutation sites to be detected from comparison results of a prover and a parent, obtaining corresponding reference sequences of all comparison results according to the comparison positions of the sequences to a reference genome, and comparing the result sequences with the reference sequences in a double sequence manner; analyzing and obtaining the corresponding position relation between all bases on each comparison sequence and the reference genome sequence according to the analysis of the double-sequence comparison result, and further confirming all normal and mutation sites on each sequencing result; recording the normal and mutation sites in all the comparison results, including the bases appearing on the recording sites and the times of the base appearance, counting the bases of all the sites, and judging the parent sources according to the following method: according to comparison results of all the mutations to be detected, respectively splicing to obtain a sequence containing the mutations to be detected of each of a prover and a parent, traversing all recorded base positions, checking whether the base positions are definitely inherited from one of the two parents, and judging that the source of the mutation site to be detected is the parent if the base of the one of the two parents is definitely inherited; if the method I can not find that any base is definitely derived from a certain party of both parents, respectively counting all comparison results which contain the site but are not mutated at the site in comparison results of the foreigners and the parents, respectively splicing to obtain a sequence which does not contain the mutation to be detected, traversing all base positions in the same way, checking whether the sites have bases definitely inherited from the certain party, if the bases definitely inherit from the certain party, judging that the sequence which is not mutated at the site in the foreigners is from the certain party, and the mutation at the site is from the other party, namely the mutation site to be detected is derived from the other party;
The new mutation judging step comprises the steps of respectively extracting all comparison results containing the site to be detected from comparison results of a prover and a parent, obtaining corresponding reference sequences of all comparison results according to the comparison positions of the sequences to a reference genome, and comparing the result sequences with the reference sequences in a double sequence manner; analyzing and obtaining the corresponding position relation between all bases on each comparison sequence and the reference genome sequence according to the analysis of the double-sequence comparison result, and further confirming all normal and mutation sites on each sequencing result; recording the normal and mutation sites in all the comparison results, including the bases appearing on the recording sites and the times of the base appearance, counting the bases of all the sites, and judging the parent sources according to the following method: the method comprises the steps of (1) splicing to obtain a sequence containing the mutation to be detected of a prover according to all comparison results containing the mutation to be detected, wherein the sequence contains the mutation to be detected of the prover, and the base combination from two chromosome chains is obtained by splicing the parent results, traversing all positions of the sequence spliced by the prover and the result obtained by splicing the parent, judging whether the base definitely originates from one of the two parents exists at the positions of the prover, and judging that the source of the new mutation site is the one if the base definitely inherits from the one of the two parents exists at the positions; in the method (2), if the method (1) cannot find that any base is definitely derived from one of the parents, splicing again from the comparison result of the prover to obtain a sequence of the prover, which does not contain the mutation to be detected, and then combining the sequence with the parent result obtained by splicing in the method (1), repeating the traversing steps in the method (1), judging whether any base of the prover on one chain which does not have the mutation is directly inherited from one of the parents, and if the base inherited from one of the parents is found, judging that the chain which does not have the mutation is derived from the other party, and judging that the chain which contains the mutation is derived from the other parent; and (3) if the method (1) and the method (2) cannot judge which of the parents comes from the newly mutated strand, carrying out reckoning on all bases of comparison results of the prover and the parents near the site according to the positions to obtain base genotypes of all the positions, searching the sites of heterozygous mutation of the prover and the parents, namely adjacent heterozygous sites, judging parent sources of the adjacent heterozygous sites by adopting a parent heterozygous site source judging step, and deducing parent sources of the newly mutated sites according to the parent sources of the adjacent heterozygous sites.
The method for judging the source of the parent heterozygote site comprises the step of performing parent source analysis according to the chain with mutation in the heterozygote mutation site, and the step of performing parent source analysis according to the chain without mutation in the heterozygote mutation site, wherein it can be understood that one of the source of the chain without mutation and the parent is judged, and the chain with mutation is necessarily sourced from the other.
In the present application, a new mutation refers to a new heterozygous mutation, and it is necessary to determine which strand is derived from a parent; and, since it is a new mutation, it means that at this site, both parents have no mutation; if the new mutation is a homozygous mutation, it can be directly determined that the source of both strands is both parents. Similarly, the method (1) in the new mutation judgment step corresponds to the method of the parent heterozygous site origin judgment step, and the parent origin analysis is directly performed on the mutated strand. The method (2) corresponds to the second method of the parent heterozygous site source judging step, and is to conduct parent source analysis on the chain which is not mutated. Searching the sites of heterozygous mutation of both a precursor and a parent near the new mutation site, so as to conveniently analyze the parent source according to the parent heterozygous site source judging step; wherein, the parent source of the new mutation site is deduced from the parent sources of adjacent heterozygous sites, e.g. the strand of the mutation in the heterozygous mutation site is identical to the strand of the mutation in the new mutation site, the deduced parent source of the heterozygous mutation is the parent source of the new mutation, whereas if the mutations are not on the same strand, the parent source of the new mutation is the other party different from the parent source of the heterozygous mutation site.
It should be noted that, the method for detecting the point mutation parent source in the application is designed separately for the complex mutation situation, that is, the situation that the foreigner and the parent are heterozygous mutations and the foreigner are new mutations, so that the point mutation parent source under the complex situation can be accurately judged.
In one implementation of the present application, the second generation sequencing alignment results include the location in the reference genome to which the sequencing results are matched, the sequencing quality, the degree of matching of reads to the reference genome sequence.
It should be noted that, in all the comparison results, in order to ensure the accuracy of the subsequent mutation source detection, the comparison results need to be pre-filtered according to some indexes. The filtering index used in the method is the read sequencing quality and the degree of comparison matching. For example, the information in the alignment results obtained from the alignment tool, i.e., the unmatched upper, second matched position, quality control failure, and repeated sequencing results, may be filtered, and may be automatically generated after the BWA tool alignment.
In one implementation of the present application, the parent heterozygous site source determining step, each position only retains more than 5 bases; similarly, in the new mutation judgment step, only the bases present at more than 5 times at each site are retained in the statistics for each sample.
It should be noted that, only bases which appear more than 5 times are reserved, so as to eliminate individual errors in the sequencing process and improve the accuracy of analysis.
In one implementation of the present application, the adjacent heterozygous site searched for in the new mutation judgment step is a heterozygous mutation within a base site range traversed by the method (1) or the method (2).
It will be appreciated that the relationship between the strand containing the primary new mutation and the adjacent heterozygous mutation, i.e. whether on the same chromosome or on a different chromosome, can only be ensured if the adjacent heterozygous mutation is within the base site range previously traversed, thereby ensuring the accuracy of the derivation of the source of the new mutation parent.
The second aspect of the application discloses a device for detecting a parent source of a point mutation, which comprises a comparison result acquisition module, a genotype analysis module, a parent source preliminary judgment module, a parent heterozygous site source judgment module and a new mutation judgment module;
the comparison result acquisition module is used for acquiring second-generation sequencing comparison results of ancestral three persons of a forerunner and a parent containing the position of the mutation site to be detected, namely, the results formed after the original second-generation sequencing results are compared with the matching positions on the reference genome;
The genotype analysis module is used for analyzing and obtaining genotypes of mutations generated by mutation sites to be detected, namely homozygosity or heterozygosity, and genotypes of parents at the sites according to second-generation sequencing comparison results;
the parent source preliminary judgment module is used for carrying out parent source preliminary judgment according to genotypes and the following rules: (1) The mutation site to be detected is homozygous mutation, and the parent sources are judged to be both parents; (2) The mutation site to be detected is heterozygous mutation, one of parents of the site has no mutation, and the other parent has mutation, so that the mutation site to be detected is derived from the mutant parent; (3) The mutation site to be detected is heterozygous mutation, one of parents of the site is homozygous mutation, the other parent is heterozygous mutation, and the mutation site to be detected is derived from one of homozygous mutation; (4) The mutation site to be detected is heterozygous mutation, and both parents at the site are heterozygous mutation, and the parent heterozygous mutation is marked as a parent heterozygous site; (5) Marking the mutation site to be detected as new mutation if both parents have no mutation at the corresponding site of the mutation site;
the parent heterozygous site source judging module is used for respectively extracting all comparison results containing the mutation site to be detected from comparison results of a prover and a parent, obtaining corresponding reference sequences of all comparison results according to the comparison positions of the sequences to a reference genome, and comparing the result sequences with the reference sequences in a double sequence manner; analyzing and obtaining the corresponding position relation between all bases on each comparison sequence and the reference genome sequence according to the analysis of the double-sequence comparison result, and further confirming all normal and mutation sites on each sequencing result; recording the normal and mutation sites in all the comparison results, including the bases appearing on the recording sites and the times of the base appearance, counting the bases of all the sites, and judging the parent sources according to the following method: according to comparison results of all the mutations to be detected, respectively splicing to obtain a sequence containing the mutations to be detected of each of a foreigner and a parent, traversing all recorded base positions, checking whether base definitely inherits from one of the two parents at the base positions, and judging that the source of the mutation site to be detected is the parent if the base definitely inherits from the one base exists; if the method I can not find that any base is definitely derived from a certain party of both parents, respectively counting all comparison results which contain the site but are not mutated at the site in comparison results of the foreigners and the parents, respectively splicing to obtain a sequence which does not contain the mutation to be detected, traversing all base positions in the same way, checking whether the sites have bases definitely inherited from the certain party, if the bases definitely inherit from the certain party, judging that the sequence which is not mutated at the site in the foreigners is from the certain party, and the mutation at the site is from the other party, namely the mutation site to be detected is derived from the other party;
The new mutation judging module is used for respectively extracting all comparison results containing the site to be detected from comparison results of a prover and a parent, obtaining corresponding reference sequences of all comparison results according to the positions of the comparison results to a reference genome, and comparing the result sequences with the reference sequences in a double sequence manner; analyzing and obtaining the corresponding position relation between all bases on each comparison sequence and the reference genome sequence according to the analysis of the double-sequence comparison result, and further confirming all normal and mutation sites on each sequencing result; recording the normal and mutation sites in all the comparison results, including the bases appearing on the recording sites and the times of the base appearance, counting the bases of all the sites, and judging the parent sources according to the following method: the method comprises the steps of (1) splicing to obtain a sequence containing the mutation to be detected of a prover according to all comparison results containing the mutation to be detected, wherein the sequence contains the mutation to be detected of the prover, and the base combination from two chromosome chains is obtained by splicing the parent results, traversing all positions of the sequence spliced by the prover and the result obtained by splicing the parent, judging whether the base definitely originates from one of the two parents exists at the positions of the prover, and judging that the source of the new mutation site is the one if the base definitely inherits from the one of the two parents exists at the positions; in the method (2), if the method (1) cannot find that any base is definitely derived from one of the parents, splicing again from the comparison result of the prover to obtain a sequence of the prover, which does not contain the mutation to be detected, and then combining the sequence with the parent result obtained by splicing in the method (1), repeating the traversing steps in the method (1), judging whether any base of the prover on one chain which does not have the mutation is directly inherited from one of the parents, and if the base inherited from one of the parents is found, judging that the chain which does not have the mutation is derived from the other party, and judging that the chain which contains the mutation is derived from the other parent; and (3) if the method (1) and the method (2) cannot judge which of the parents comes from the newly mutated strand, carrying out reckoning on all bases of comparison results of the prover and the parents near the site according to the positions to obtain base genotypes of all the positions, searching the sites of heterozygous mutation of the prover and the parents, namely adjacent heterozygous sites, judging parent sources of the adjacent heterozygous sites by adopting a parent heterozygous site source judging module, and deducing parent sources of the newly mutated sites according to the parent sources of the adjacent heterozygous sites.
It should be noted that, the device for detecting the point mutation parent source in the present application is actually a method for realizing the point mutation parent source in the present application through each module respectively; thus, specific definition of each module may be found in reference to each corresponding step in the methods of detecting point mutation parent sources herein, and will not be described in detail herein.
A third aspect of the present application discloses an apparatus for detecting a parent source of a point mutation, the apparatus comprising a memory and a processor; a memory including a memory for storing a program; a processor comprising means for implementing the parent source of the point mutation of the present application by executing a program stored in a memory.
A fourth aspect of the present application discloses a computer readable storage medium having stored therein a program executable by a processor to implement a method of detecting point mutation parent sources of the present application.
Due to the adoption of the technical scheme, the beneficial effects of the application are that:
the method for detecting the point mutation parent source not only can detect the parent source of the heterozygous mutation of the forerunner who is heterozygous mutation on both parents, but also can detect the new mutation source of the forerunner; in addition, the method is based on the original second-generation sequencing result for analysis, so that the accuracy of the parent source detection result is greatly improved.
Drawings
FIG. 1 is a flow diagram of a method of detecting a point mutation parent source in an embodiment of the present application;
FIG. 2 is a block diagram of an apparatus for detecting a parent source of a point mutation in an embodiment of the present application;
FIG. 3 is a schematic representation of a mutant form that may be directly judged to be of parent origin in an embodiment of the present application;
FIG. 4 is a schematic representation of a mutant form identified by heterozygous mutant source chains in the examples of the present application;
FIG. 5 is a schematic representation of a mutant form identified by a new mutant source strand in the examples of the present application;
FIG. 6 is a graph showing the comparison of a heterozygous mutant-derived parent in the examples of the present application;
FIG. 7 is a diagram showing the results of comparison of the source judgment examples of the parent chain in which a new mutation is located in the examples of the present application.
Detailed Description
The present application is described in further detail below with reference to the accompanying drawings by way of specific embodiments. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present application. However, those skilled in the art will readily recognize that some of the features may be omitted, or substituted for other devices, materials, or methods in different situations. In some instances, some operations associated with the present application have not been shown or described in the specification to avoid obscuring the core portions of the present application, and may not be necessary for a person skilled in the art to describe in detail the relevant operations based on the description herein and the general knowledge of one skilled in the art.
In the judgment of the source strand of the mutation, it is actually judged on which chromosome the mutation is inherited from the parent. For mutations that exist only in one party, the source can be directly determined. However, in practical judgment, many cases are complicated, for example, the first-person and the parent are both heterozygous mutations, the first-person has new mutations, and the like, and the corresponding judgment logic needs to be set in the cases to judge the source chain. The method utilizes the result of second generation sequencing to judge the source chromosome of the point mutation, thereby providing more effective information for clinical diagnosis.
The core principle used in the present application is based on comparison between all the comparison results obtained by sequencing of the precursor and the parent, which contain the mutation, and the base differences at all the corresponding positions of the comparison results within the range are utilized, so that the genetic source of the chromosome chain containing the mutation can be deduced or reversely deduced from the side. The specific base logic for determining any base or mutation is as follows: for the site of any heterozygous mutation, if one parent contains the mutation and the other is not, then the mutation is highly likely to be from the parent who has the mutation; if one parent is homozygous for a mutation and the other is heterozygous, then there is a high probability that the mutation will be from the parent who contains the homozygous mutation. When a certain base site of the foreigner is not mutated, if one of parents is heterozygous, the parent can only inherit one chromosome with the site not mutated to the foreigner; when a certain base site of the forensic person is homozygous mutation, if one of parents is heterozygous mutation, the parent can only inherit the chain with mutation at the base site to the forensic person.
According to the judgment basis, for the point mutation site to be judged, the mutation condition of the current site cannot directly judge the mutation source of the precursor, so that the source chain of the mutation to be judged can be determined according to the source of other bases on the same sequence by starting from other base sites of the sequencing reading section containing the mutation site, judging which base on the other sites comes from a parent. In order to directly analyze other sites with definite positions relative to the target mutation (i.e., located on the same strand or on different strands), the present application directly analyzes sequencing results, in which each sequencing result is from the same chromosome, and the strand from the same chromosome is generally inherited from a parent, so that the source of the strand can be determined by using the sources of some specific mutations or bases in the strand from the same chromosome, and thus the parent strand from which the target mutation is derived is known. In the judging flow, in order to further increase the detection efficiency and accuracy, the sequence with mutation at the site and the sequence without mutation need to be separately judged, and the advantage of the separate judgment is that the two aspects can be started, namely, the side from which the chain with mutation at the site comes or the side from which the chain without mutation at the site comes can be analyzed. Based on the analysis results of the two cases, the parent of the target mutation source can be judged according to the genetic relationship between the chains which are not mutated and the mutated chains.
In the design of the application, all comparison results covering the mutation positions to be detected are extracted according to the positions of the mutations to be verified based on comparison files obtained by second-generation sequencing from three families, and the base distribution of three families on the fragment is compared according to the analysis and treatment of the comparison results, so that the judgment basis of the parent of the mutation source can be obtained.
Based on the above researches and knowledge, the application creatively provides a method for detecting a parent source of a point mutation, which comprises a comparison result acquisition step 11, a genotype analysis step 12, a parent source preliminary judgment step 13, a parent heterozygous site source judgment step 14 and a new mutation judgment step 15 as shown in fig. 1.
The comparison result obtaining step 11 includes obtaining second-generation sequencing comparison results of three families of a forerunner and a parent containing the position of the mutation site to be detected, namely, the results obtained after the original second-generation sequencing results are compared with the matching positions on the reference genome.
For example, the second generation sequencing comparison result records information such as the position of a certain segment of sequencing result matched with the reference genome, the sequencing quality, whether the read and the reference sequence are completely matched, and the like. In the conventional procedure, the comparison result can be obtained by comparison with a BWA (Burrows-Wheeler-Alignment Tool) Tool based on the original second-generation sequencing result, and BWA is software dedicated to comparing the second-generation sequencing result to a matching position on a reference genome, which can match all reads of the second-generation sequencing to the reference sequence closest to the reads through comparison, so that mutation information on a chromosome can be analyzed according to the result after comparison. Meanwhile, the mutation position to be detected and the bases before and after the mutation at the position are required to be obtained, and if all the point mutations in the whole genome are treated, all the point mutations are only required to be treated in a circulating way. The information of point mutations can be detected by other trust tools, such as GATK (Genome Analyse tool kit), GATK, which is a set of software used to process high-throughput sequencing data, and is mainly capable of analyzing the point mutations and INDEL in human sequencing data, and GATK is a gold standard for human genome mutation detection because of the accuracy and powerful functions of all the point mutations obtained by analyzing the comparison results.
Genotype analysis step 12, which comprises obtaining the genotype of the mutation site to be detected, namely homozygosity or heterozygosity, and the genotypes of the parents at the site according to the analysis of the second-generation sequencing comparison result. The mutation genotypes of both the precursor and parent, i.e., homozygous or heterozygous, can be directly determined as to whether the source strand of the mutation site is available.
A parent source preliminary judgment step 13, which comprises the steps of carrying out parent source preliminary judgment according to genotypes and the following rules: (1) If the mutation site to be detected is homozygous mutation, judging that the parent sources are both parents, for example, the A diagram of FIG. 3; (2) The mutation site to be detected is heterozygous mutation, one of parents of the site has no mutation, and the other parent has mutation, so that the mutation site to be detected is derived from the side with mutation, for example, a diagram B of FIG. 3; (3) The mutation site to be detected is heterozygous mutation, one of parents of the site is homozygous mutation, and the other parent is heterozygous mutation, and the mutation site to be detected is derived from one side of homozygous mutation, for example, a graph C of FIG. 3; (4) The mutation site to be detected is heterozygous mutation, and both parents at the site are heterozygous mutation, and the parent heterozygous mutation is marked as a parent heterozygous site; (5) And marking the mutant as new mutation if no mutation exists in the parent and the corresponding site of the mutation site to be detected. In FIG. 3, the lines represent chromosomal sequences, the bars represent mutations at that position, and the a mutation is the mutation site to be detected.
A parent heterozygous site source judging step 14, which comprises respectively extracting all comparison results containing mutation sites to be detected from comparison results of a prover and a parent, obtaining corresponding reference sequences of all comparison results according to the comparison positions of the sequences to a reference genome, and comparing the result sequences with the reference sequences in a double sequence manner; analyzing and obtaining the corresponding position relation between all bases on each comparison sequence and the reference genome sequence according to the analysis of the double-sequence comparison result, and further confirming all normal and mutation sites on each sequencing result; recording the normal and mutation sites in all the comparison results, including the bases appearing on the recording sites and the times of the base appearance, counting the bases of all the sites, and judging the parent sources according to the following method: according to comparison results of all the mutations to be detected, respectively splicing to obtain a sequence containing the mutations to be detected of each of a foreigner and a parent, traversing all recorded base positions, checking whether base definitely inherits from one of the two parents at the base positions, and judging that the source of the mutation site to be detected is the parent if the base definitely inherits from the one base exists; if the method I can not find that any base is definitely derived from a certain party of both parents, respectively counting all comparison results of the foreigners and the parents, which contain the site but are not mutated at the site, respectively splicing to obtain a sequence which does not contain the mutation to be detected, traversing all base positions similarly, checking whether the sites have bases definitely inherited from the certain party, and if the bases definitely inherit from the certain party, judging that the sequence which is not mutated at the site in the foreigners is derived from the certain party, and the mutation at the site is derived from the other party, namely the mutation site to be detected is derived from the other party.
In the application, if the three families are heterozygous mutations at the locus, all the comparison results containing the mutation locus (the locus is mutated) are extracted from the comparison results of the prover and the parents respectively, and all the sequences in the comparison results are recorded according to the comparison of the sequences to the corresponding positions of the reference genome, namely, the bases appearing at a certain locus are recorded, and the bases appear several times. To exclude individual errors occurring during sequencing, only more than 5 bases were eventually retained at each position. After counting the bases at all sites, since only the sequencing result of the mutation at the detection site is counted, theoretically, the results are only from the same chromosome of each sample, and thus, a sequence containing the mutation of each of the precursor and the parent can be spliced. The base specifically inherited from one side may be a mutation or a wild base, and if the base specifically inherited from one side exists, the source of the site to be detected may be determined as that side. In the specific cases of the first method, for example, A, B, C in fig. 4, in the a, B and C diagrams, the sequence with a frame is a sequence obtained by splicing the alignment results containing the target mutation, i.e., the mutation at the a site, and only the chain needs to be analyzed in the judgment of the mutation source in this step, and only the chain is considered in the next judgment process. In the case of panel A, there is a b mutation near the a mutation, and the b mutation is obviously from the father, so it can be judged that the a mutation is also derived from the father's strand as is the b mutation. In the case of panel B there is a B site near the a mutation, which is not mutated, so this unmutated base inherits from the father, and it is deduced that the a mutation also inherits from the father as does the base with the unmutated B site. In the case of panel C, a mutation occurs at position b which can only inherit the mutated strand from the father, thus judging that both mutations at position a and position b inherit the strand from the father.
If the method one can not find that any base is definitely derived from a certain party, then respectively carrying out the same treatment on all comparison results which contain the site but are not mutated at the site in the comparison results of the foreigners and parents, respectively splicing to obtain a sequence which does not contain the mutation to be detected, traversing all base positions, checking whether the sites have bases definitely inherited from a certain party, if the bases definitely inherit from a certain party, judging that the sequence which is not mutated at the site in the foreigners is from the party, and the mutation at the site is from the other party, namely the method two. In the second method, the sequence that does not include the target mutation is used for judgment, for example, in the case of D, E in fig. 4, in the case of D and E, the sequence with a frame is a sequence obtained by splicing the comparison results of three samples, namely, the a site is not mutated, the mutation source is judged in the step, only the chain is analyzed, and only the chain is considered in the next judgment process. In the case of panel D, the prover has a mutation at position b and this mutation is clearly inherited from the father, thus judging that the strand of the prover that is not mutated at position a is from the father, and deducing that the strand of the prover that is mutated at position a is from the mother. In the case of panel E, no mutation occurs at position b and this unmutated base can only be inherited from the mother, thus it is inferred that the unmutated strand at position b in the precursor comes from the mother and the mutated strands at positions b and a are inherited from the father.
A new mutation judging step 15, which comprises the steps of respectively extracting all comparison results containing the site to be detected from comparison results of a prover and a parent, obtaining corresponding reference sequences of all comparison results according to the comparison positions of the sequences to a reference genome, and comparing the result sequences with the reference sequences in a double sequence manner; analyzing and obtaining the corresponding position relation between all bases on each comparison sequence and the reference genome sequence according to the analysis of the double-sequence comparison result, and further confirming all normal and mutation sites on each sequencing result; recording the normal and mutation sites in all the comparison results, including the bases appearing on the recording sites and the times of the base appearance, counting the bases of all the sites, and judging the parent sources according to the following method: the method comprises the steps of (1) splicing to obtain a sequence containing the mutation to be detected of a prover according to all comparison results containing the mutation to be detected, wherein the sequence contains the mutation to be detected of the prover, and the base combination from two chromosome chains is obtained by splicing the parent results, traversing all positions of the sequence spliced by the prover and the result obtained by splicing the parent, judging whether the base definitely originates from one of the two parents exists at the positions of the prover, and judging that the source of the new mutation site is the one if the base definitely inherits from the one of the two parents exists at the positions; in the method (2), if the method (1) cannot find that any base is definitely derived from one of the parents, splicing again from the comparison result of the prover to obtain a sequence of the prover, which does not contain the mutation to be detected, and then combining the sequence with the parent result obtained by splicing in the method (1), repeating the traversing steps in the method (1), judging whether any base of the prover on one chain which does not have the mutation is directly inherited from one of the parents, and if the base inherited from one of the parents is found, judging that the chain which does not have the mutation is derived from the other party, and judging that the chain which contains the mutation is derived from the other parent; and (3) if the method (1) and the method (2) cannot judge which of the parents comes from the newly mutated strand, carrying out reckoning on all bases of comparison results of the prover and the parents near the site according to the positions to obtain base genotypes of all the positions, searching the sites of heterozygous mutation of the prover and the parents, namely adjacent heterozygous sites, judging parent sources of the adjacent heterozygous sites by adopting a parent heterozygous site source judging step, and deducing parent sources of the newly mutated sites according to the parent sources of the adjacent heterozygous sites.
In the present application, a new mutation means a mutation that is not inherited from a parent but is inherited from a chromosome of a parent at a position where the mutation occurs, and therefore it is only possible to determine on which chromosome the mutation is inherited. If the site is new mutation, the mutation site can be extracted from the comparison result of the precursor, all the comparison results of the mutation of the base at the site are recorded, and all the bases appearing in the sequences are recorded at the positions of the reference genome. Unlike the determination of heterozygous mutation, since the site to be verified is new, no mutation occurs in this site in the parent, and thus all sequencing results containing this site, i.e., base combinations from both strands of the chromosome, are extracted from the parent's alignment and the bases present at each position are recorded. To exclude individual errors occurring during sequencing, only more than 5 bases at each site were retained in the statistics for each sample.
In the judging process, traversing all the positions counted by the prover and the parent, judging whether the prover can directly judge the base which is definitely derived from a certain parent in the positions, and if the base which is directly inherited from a certain parent can be found in the traversing process, directly judging the source of the site to be verified as the certain parent, namely the method (1). In the case of method (1), for example, in both cases A and B in FIG. 5, the boxed gray region sequences in both cases A and B are the results of extraction from the ancestor and parent, respectively, where the target site a is not mutated, and thus for either position, the base combinations from the corresponding positions of the two chromosomes, the parent cannot obtain the exact sequence on this fragment. As with the heterozygous mutation judgment method above, only the bases in the framed gray region were examined. In the case of panel a, since there is a mutation at the b position near a, and only the parent b position detects the mutation, it is judged that the b mutation is from the parent, and thus the a mutation occurs on the chromosome from the parent. In the case of panel B, the mutation at position B is a heterozygous mutation, and therefore the mutation at position B can only be derived from the side containing the homozygous mutation at position B, i.e., the genetic origin is from the father, and therefore it is judged that the mutation at position a occurs on the chromosome from the father.
If a base that can be directly determined from the sequence that contains the mutation in the prover cannot be found, i.e., the parent source cannot be determined by the method (1), then all the comparison results that do not have the mutation at that site are again extracted from the comparison results of the prover, and the bases that appear at each site in these results are recorded as well, then the steps of traversing the above determination are repeated to determine whether any base on one strand that does not have the mutation in the prover is directly inherited from one parent, and if a base inherited from one parent is found, the strand that does not have the mutation is determined to be from that parent, and the strand that contains the mutation is determined to be from the other parent, i.e., the method (2). In the method (2), for example, in case C of FIG. 5, the grey part of the frame in the C diagram is the result of extraction from the precursor and the parent respectively, and as in the A diagram and the B diagram, the sequence of the parent cannot obtain the exact sequence and is therefore the base combination from the corresponding positions of the two chromosomes, the precursor in the C diagram is mutated at the B position, only the mother has the mutation, and therefore the sequence with the mutation at the B position is inherited from the mother, and thus the sequence with the non-mutation at the B position is inversely deduced to be inherited from the father, so that the a mutation is judged to be also generated on the chain inherited from the father.
If the method (1) and the method (2) cannot judge from which side the new mutation occurs, reckoning all bases of comparison results of a prover and a parent near the locus according to the positions, obtaining base genotypes of all positions, searching loci of the parent in all positions, wherein the three loci are heterozygous mutations, if the loci meet the condition, judging the heterozygous mutation source according to the method for judging the heterozygous mutation source, and accordingly pushing the new mutation source chain reversely, namely the method (3). In the case of method (3) such as D in FIG. 5, if either method (1) or method (2) is used in the case of D, the parent can only judge that the b and c sites are heterozygous mutations because the parent only has a base combination at each site, and thus cannot judge the parent strand of the chromosome in which the a mutation is located. If the heterozygous mutation at the b position is used, then the chain containing the b mutation in the foreigner can be judged to be inherited from the father according to the judging method of the heterozygous mutation source, namely the parent heterozygous site source judging step, so that the a mutation is also located on the chromosome from the father. It should be noted that when judging from adjacent heterozygous mutation sites, it is necessary to ensure that the heterozygous mutation is within the base site that was traversed before, so that the relationship between the strand containing the original mutation and the heterozygous mutation (i.e., whether on the same chromosome or on a different chromosome) can be directly ensured, and thus the source of the mutation can be deduced correctly.
Those skilled in the art will appreciate that all or part of the functions of the above-described methods may be implemented by means of hardware, or by means of a computer program. When all or part of the functions of the above method are implemented by means of a computer program, the program may be stored in a computer readable storage medium, and the storage medium may include: read-only memory, random access memory, magnetic disk, optical disk, hard disk, etc., and the program is executed by a computer to realize the above-mentioned 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 functions described above can be realized. In addition, when all or part of the functions in the above embodiments are implemented by means of a computer program, the program may be stored in a storage medium such as a server, another computer, a magnetic disk, an optical disk, a flash disk, or a removable hard disk, and all or part of the functions in the above methods may be implemented by downloading or copying the program into a memory of a local device or updating a version of a system of the local device, and executing the program in the memory by a processor.
Therefore, based on the method for detecting the parent source of the point mutation in the present application, the present application proposes a device for detecting the parent source of the point mutation, as shown in fig. 2, which includes a comparison result obtaining module 21, a genotype analyzing module 22, a parent source preliminary judging module 23, a parent heterozygous site source judging module 24 and a new mutation judging module 25.
The comparison result obtaining module 21 is used for obtaining second-generation sequencing comparison results of three families of a forerunner and a parent containing the mutation site to be detected, namely, results formed by comparing the original second-generation sequencing results with matching positions on a reference genome.
The genotype analysis module 22 is used for analyzing and obtaining the genotype of the mutation at the mutation site to be detected according to the second-generation sequencing comparison result, namely homozygosity or heterozygosity, and the genotypes of the parents at the site.
The parent source preliminary judgment module 23 includes a module for performing a parent source preliminary judgment according to the genotype and the following rules: (1) The mutation site to be detected is homozygous mutation, and the parent sources are judged to be both parents; (2) The mutation site to be detected is heterozygous mutation, one of parents of the site has no mutation, and the other parent has mutation, so that the mutation site to be detected is derived from the mutant parent; (3) The mutation site to be detected is heterozygous mutation, one of parents of the site is homozygous mutation, the other parent is heterozygous mutation, and the mutation site to be detected is derived from one of homozygous mutation; (4) The mutation site to be detected is heterozygous mutation, and both parents at the site are heterozygous mutation, and the parent heterozygous mutation is marked as a parent heterozygous site; (5) And marking the mutant as new mutation if no mutation exists in the parent and the corresponding site of the mutation site to be detected.
The parent heterozygous site source judging module 24 is used for respectively extracting all comparison results containing the mutation site to be detected from comparison results of a prover and a parent, and obtaining corresponding reference sequences of all the comparison results according to the positions of the comparison results to a reference genome and comparing the result sequences with the reference sequences in a double sequence manner; analyzing and obtaining the corresponding position relation between all bases on each comparison sequence and the reference genome sequence according to the analysis of the double-sequence comparison result, and further confirming all normal and mutation sites on each sequencing result; recording the normal and mutation sites in all the comparison results, including recording the base appearing on the site and the number of times the base appears, counting the base of all the sites, and judging the parent source according to the following method: according to comparison results of all the mutations to be detected, respectively splicing to obtain a sequence containing the mutations to be detected of each of a foreigner and a parent, traversing all recorded base positions, checking whether base definitely inherits from one of the two parents at the base positions, and judging that the source of the mutation site to be detected is the parent if the base definitely inherits from the one base exists; if the method I can not find that any base is definitely derived from a certain party of both parents, respectively counting all comparison results of the foreigners and the parents, which contain the site but are not mutated at the site, respectively splicing to obtain a sequence which does not contain the mutation to be detected, traversing all base positions similarly, checking whether the sites have bases definitely inherited from the certain party, and if the bases definitely inherit from the certain party, judging that the sequence which is not mutated at the site in the foreigners is derived from the certain party, and the mutation at the site is derived from the other party, namely the mutation site to be detected is derived from the other party.
The new mutation judging module 25 is configured to extract all comparison results including the site to be tested from comparison results of the prover and the parent, respectively, obtain corresponding reference sequences for sequences of all comparison results according to the positions of the comparison results to the reference genome, and perform double-sequence comparison on the result sequences and the reference sequences; analyzing and obtaining the corresponding position relation between all bases on each comparison result sequence and the reference genome sequence according to the analysis of the double-sequence comparison result, and further confirming all normal and mutation sites on each sequencing result; recording the normal and mutation sites in all the comparison results, including recording the base appearing on the site and the number of times the base appears, counting the base of all the sites, and judging the parent source according to the following method: the method comprises the steps of (1) splicing to obtain a sequence containing the mutation to be detected of a prover according to all comparison results containing the mutation to be detected, wherein the sequence contains the mutation to be detected of the prover, and the base combination from two chromosome chains is obtained by splicing the parent results, traversing all positions of the sequence spliced by the prover and the result obtained by splicing the parent, judging whether the base definitely originates from one of the two parents exists at the positions of the prover, and judging that the source of the new mutation site is the one if the base definitely inherits from the one of the two parents exists at the positions; in the method (2), if the method (1) cannot find that any base is definitely derived from one of the parents, re-splicing to obtain a sequence of a prover which does not contain the mutation to be detected, combining the sequence with the parent result obtained by splicing in the method (1), repeating the traversing steps in the method (1), judging whether any base of the prover on one chain which does not have the mutation is directly inherited from one of the parents, and if the base inherited from one of the parents is found, judging that the chain which does not have the mutation is derived from the other one of the parents and the chain which contains the mutation is derived from the other one of the parents; and (3) if the method (1) and the method (2) cannot judge which of the parents comes from the newly mutated strand, carrying out reckoning on all bases of comparison results of the prover and the parents near the site according to the positions to obtain base genotypes of all the positions, searching the sites of heterozygous mutation of the prover and the parents, namely adjacent heterozygous sites, judging parent sources of the adjacent heterozygous sites by adopting a parent heterozygous site source judging module, and deducing parent sources of the newly mutated sites according to the parent sources of the adjacent heterozygous sites.
There is also provided in another implementation of the present application an apparatus for detecting a parent source of a point mutation, the apparatus comprising a memory and a processor; a memory including a memory for storing a program; a processor comprising a program for implementing the following method by executing a program stored in a memory: the comparison result obtaining step comprises the step of obtaining second-generation sequencing comparison results of ancestral three persons of a forerunner and a parent containing the position of the mutation site to be detected, namely, the result formed by comparing the original second-generation sequencing results to the matching position on the reference genome; genotype analysis, namely, analyzing the genotype of mutation at a mutation site to be detected, namely, homozygosity or heterozygosity, and the genotypes of parents at the site according to a second-generation sequencing comparison result; the preliminary parent source judging step comprises the steps of carrying out preliminary parent source judging according to genotypes and the following rules: (1) The mutation site to be detected is homozygous mutation, and the parent sources are judged to be both parents; (2) The mutation site to be detected is heterozygous mutation, one of parents of the site has no mutation, and the other parent has mutation, so that the mutation site to be detected is derived from the mutant parent; (3) The mutation site to be detected is heterozygous mutation, one of parents of the site is homozygous mutation, the other parent is heterozygous mutation, and the mutation site to be detected is derived from one of homozygous mutation; (4) The mutation site to be detected is heterozygous mutation, and both parents at the site are heterozygous mutation, and the parent heterozygous mutation is marked as a parent heterozygous site; (5) Marking the mutation site to be detected as new mutation if both parents have no mutation at the corresponding site of the mutation site; the parent heterozygous site source judging step comprises the steps of respectively extracting all comparison results containing mutation sites to be detected from comparison results of a prover and a parent, obtaining corresponding reference sequences of all comparison results according to the comparison positions of the sequences to a reference genome, and comparing the result sequences with the reference sequences in a double sequence manner; analyzing and obtaining the corresponding position relation between all bases on each comparison sequence and the reference genome sequence according to the analysis of the double-sequence comparison result, and further confirming all normal and mutation sites on each sequencing result; recording the normal and mutation sites in all the comparison results, including recording the base appearing on the site and the number of times the base appears, counting the base of all the sites, and judging the parent source according to the following method: according to comparison results of all the mutations to be detected, respectively splicing to obtain a sequence containing the mutations to be detected of each of a foreigner and a parent, traversing all recorded base positions, checking whether base definitely inherits from one of the two parents at the base positions, and judging that the source of the mutation site to be detected is the parent if the base definitely inherits from the one base exists; if the method I can not find that any base is definitely derived from a certain party of both parents, respectively counting all comparison results which contain the site but are not mutated at the site in comparison results of the foreigners and the parents, respectively splicing to obtain a sequence which does not contain the mutation to be detected, traversing all base positions in the same way, checking whether the sites have bases definitely inherited from the certain party, if the bases definitely inherit from the certain party, judging that the sequence which is not mutated at the site in the foreigners is from the certain party, and the mutation at the site is from the other party, namely the mutation site to be detected is derived from the other party; the new mutation judging step comprises the steps of extracting all comparison results containing the site to be detected from comparison results of a precursor, obtaining corresponding reference sequences of all comparison results according to the comparison positions of the sequences to a reference genome, and comparing the result sequences with the reference sequences in a double sequence manner; analyzing and obtaining the corresponding position relation between all bases on each comparison sequence and the reference genome sequence according to the analysis of the double-sequence comparison result, and further confirming all normal and mutation sites on each sequencing result; recording the normal and mutation sites in all the comparison results, including recording the base appearing on the site and the number of times the base appears, counting the base of all the sites, and judging the parent source according to the following method: the method comprises the steps of (1) splicing to obtain a sequence containing the mutation to be detected of a prover according to all comparison results containing the mutation to be detected, wherein the sequence contains the mutation to be detected of the prover, and the base combination from two chromosome chains is obtained by splicing the parent results, traversing all positions of the sequence spliced by the prover and the result obtained by splicing the parent, judging whether the base definitely originates from one of the two parents exists at the positions of the prover, and judging that the source of the new mutation site is the one if the base definitely inherits from the one of the two parents exists at the positions; in the method (2), if the method (1) cannot find that any base is definitely derived from one of the parents, re-splicing to obtain a sequence of a prover which does not contain the mutation to be detected, combining the sequence with the parent result obtained by splicing in the method (1), repeating the traversing steps in the method (1), judging whether any base of the prover on one chain which does not have the mutation is directly inherited from one of the parents, and if the base inherited from one of the parents is found, judging that the chain which does not have the mutation is derived from the other one of the parents and the chain which contains the mutation is derived from the other one of the parents; and (3) if the method (1) and the method (2) cannot judge which of the parents comes from the newly mutated strand, carrying out reckoning on all bases of comparison results of the prover and the parents near the site according to the positions to obtain base genotypes of all the positions, searching the sites of heterozygous mutation of the prover and the parents, namely adjacent heterozygous sites, judging parent sources of the adjacent heterozygous sites by adopting a parent heterozygous site source judging step, and deducing parent sources of the newly mutated sites according to the parent sources of the adjacent heterozygous sites.
There is also provided in another implementation of the present application a computer readable storage medium including a program executable by a processor to implement a method of: the comparison result obtaining step comprises the step of obtaining second-generation sequencing comparison results of ancestral three persons of a forerunner and a parent containing the position of the mutation site to be detected, namely, the result formed by comparing the original second-generation sequencing results to the matching position on the reference genome; genotype analysis, namely, analyzing the genotype of mutation at a mutation site to be detected, namely, homozygosity or heterozygosity, and the genotypes of parents at the site according to a second-generation sequencing comparison result; the preliminary parent source judging step comprises the steps of carrying out preliminary parent source judging according to genotypes and the following rules: (1) The mutation site to be detected is homozygous mutation, and the parent sources are judged to be both parents; (2) The mutation site to be detected is heterozygous mutation, one of parents of the site has no mutation, and the other parent has mutation, so that the mutation site to be detected is derived from the mutant parent; (3) The mutation site to be detected is heterozygous mutation, one of parents of the site is homozygous mutation, the other parent is heterozygous mutation, and the mutation site to be detected is derived from one of homozygous mutation; (4) The mutation site to be detected is heterozygous mutation, and both parents at the site are heterozygous mutation, and the parent heterozygous mutation is marked as a parent heterozygous site; (5) Marking the mutation site to be detected as new mutation if both parents have no mutation at the corresponding site of the mutation site; the parent heterozygous site source judging step comprises the steps of respectively extracting all comparison results containing mutation sites to be detected from comparison results of a prover and a parent, obtaining corresponding reference sequences of all comparison results according to the comparison positions of the sequences to a reference genome, and comparing the result sequences with the reference sequences in a double sequence manner; analyzing and obtaining the corresponding position relation between all bases on each comparison sequence and the reference genome sequence according to the analysis of the double-sequence comparison result, and further confirming all normal and mutation sites on each sequencing result; recording the normal and mutation sites in all the comparison results, including recording the base appearing on the site and the number of times the base appears, counting the base of all the sites, and judging the parent source according to the following method: according to comparison results of all the mutations to be detected, respectively splicing to obtain a sequence containing the mutations to be detected of each of a foreigner and a parent, traversing all recorded base positions, checking whether base definitely inherits from one of the two parents at the base positions, and judging that the source of the mutation site to be detected is the parent if the base definitely inherits from the one base exists; if the method I can not find that any base is definitely derived from a certain party of both parents, respectively counting all comparison results which contain the site but are not mutated at the site in comparison results of the foreigners and the parents, respectively splicing to obtain a sequence which does not contain the mutation to be detected, traversing all base positions in the same way, checking whether the sites have bases definitely inherited from the certain party, if the bases definitely inherit from the certain party, judging that the sequence which is not mutated at the site in the foreigners is from the certain party, and the mutation at the site is from the other party, namely the mutation site to be detected is derived from the other party; the new mutation judging step comprises the steps of extracting all comparison results containing the site to be detected from comparison results of a precursor, obtaining corresponding reference sequences of all comparison results according to the comparison positions of the sequences to a reference genome, and comparing the result sequences with the reference sequences in a double sequence manner; analyzing and obtaining the corresponding position relation between all bases on each comparison sequence and the reference genome sequence according to the analysis of the double-sequence comparison result, and further confirming all normal and mutation sites on each sequencing result; recording the normal and mutation sites in all the comparison results, including recording the base appearing on the site and the number of times the base appears, counting the base of all the sites, and judging the parent source according to the following method: the method comprises the steps of (1) splicing to obtain a sequence containing the mutation to be detected of a prover according to all comparison results containing the mutation to be detected, wherein the sequence contains the mutation to be detected of the prover, and the base combination from two chromosome chains is obtained by splicing the parent results, traversing all positions of the sequence spliced by the prover and the result obtained by splicing the parent, judging whether the base definitely originates from one of the two parents exists at the positions of the prover, and judging that the source of the new mutation site is the one if the base definitely inherits from the one of the two parents exists at the positions; in the method (2), if the method (1) cannot find that any base is definitely derived from one of the parents, re-splicing to obtain a sequence of a prover which does not contain the mutation to be detected, combining the sequence with the parent result obtained by splicing in the method (1), repeating the traversing steps in the method (1), judging whether any base of the prover on one chain which does not have the mutation is directly inherited from one of the parents, and if the base inherited from one of the parents is found, judging that the chain which does not have the mutation is derived from the other one of the parents and the chain which contains the mutation is derived from the other one of the parents; and (3) if the method (1) and the method (2) cannot judge which of the parents comes from the newly mutated strand, carrying out reckoning on all bases of comparison results of the prover and the parents near the site according to the positions to obtain base genotypes of all the positions, searching the sites of heterozygous mutation of the prover and the parents, namely adjacent heterozygous sites, judging parent sources of the adjacent heterozygous sites by adopting a parent heterozygous site source judging step, and deducing parent sources of the newly mutated sites according to the parent sources of the adjacent heterozygous sites.
The methods and apparatus of the present application enable the determination of the parent source of a chromosome containing a point mutation in the human genome, and can be used to analyze the genetic source of the point mutation, or to analyze what side of the chromosome the point mutation is inherited from. The core principle of the application is to judge the source chain of the mutation by analyzing whether other sites except the target site can be used as the basis for judging the source chain according to the comparison between the second generation sequencing comparison results of the target site in the standard family. Specifically two different analysis cases in heterozygous and new mutations:
1. when the heterozygous mutation of the family is analyzed, whether other bases exist on the chain with mutation and the chain without mutation in the family is analyzed respectively, so that the judgment can be assisted.
2. When new mutation is analyzed, whether other bases exist on the chain with mutation and the chain without mutation of the prover can be judged in an auxiliary mode or not is analyzed, if not, other family heterozygous mutation sites are tried to be found, and auxiliary judgment is carried out by utilizing a family heterozygous mutation judging process.
Compared with the prior art, the method and the device for detecting the point mutation parent source have the following advantages:
1. The method can be used for judging heterozygous mutation sources and new mutation source chains, and the result is from a sequencing reading obtained by second generation sequencing, so that the judgment result is quite accurate because the result is actually judgment logic made according to the sequencing result of the same chromosome in the sequencing original result.
2. The judgment of the chain where the mutation is located in the application is confirmed according to a sequence in a shorter range, and the probability of homologous exchange of chromosomes of both parents in the shorter range is relatively low. The method avoids the error of judgment results caused by homologous exchange of fragments of parents in the process of analyzing the whole chromosome source.
3. The method is suitable for judging the source chain of the point mutation obtained from the NGS detection result, and can obtain more mutation information based on the existing mutation detection, thereby providing more effective information for diagnosis and treatment of genetic diseases.
It can be appreciated that the scheme of the application is not only suitable for judging the chromosome source parents containing mutation in the second generation sequencing result, but also can be used for judging certain mutation source chains by expanding the method into other sequencing technologies with longer single sequencing fragments, so that the effect of judging the chromosome of the mutation source can be enhanced. In the determination of the origin of chromosomes including mutations, the integrity of a longer chromosome fragment may be determined, but in the determination of a longer fragment, both parents may have homologous exchanges between chromosomes during meiosis, and thus the fragment may come from both parents, resulting in a certain error in the result. In addition, the methods and apparatus of the present application are also applicable to mutation-derived strand judgment in other diploid organisms.
The present application is described in further detail below by way of specific experiments. The following experiments are merely further illustrative of the present application and should not be construed as limiting the present application.
Test 1
In one example, the ancestry member was sequenced and analyzed for genetic disease using a second generation sequencing technique for disease cause diagnosis, since the ancestor may have hemipyramidal disease. After processing the second generation sequencing data, based on the mutation detection results of the GATK, it was found that the precursor had a heterozygous mutation of chr16:30097630:C > T, and that the mutation at this site could be associated with a disease, and for determining the source of this mutation, a parental source analysis was performed on the mutation according to the method for detecting the parental source of the point mutation shown in FIG. 1, as described above. In the determination of the heterozygous mutation source chain, it was found that both the ancestor and the parent in the family had heterozygous mutation at this site, based on the detection result of the mutation detection software GATK. The alignment of all the alignment results from three families containing the chr16:30097630:C > T mutation corresponding to the results is shown in FIG. 6, and it can be seen that the heterozygous mutation is present in both the precursor and parent, so that the parent of the mutant chain cannot be analyzed by the information of the site alone.
By using the method for detecting the parent source of the point mutation, all sequencing results of family members including target sites are extracted according to matching positions recorded in comparison results, and problematic comparison results are filtered according to the filtering method after the comparison results are extracted. And extracting corresponding reference sequences from the rest comparison results on the reference genome according to the comparison positions, carrying out double-sequence comparison, and confirming all normal and mutation sites on each sequencing read according to the sequence comparison results. Then classifying, merging and counting all comparison results according to six types of the prior evidence + including the mutation to be tested, the prior evidence + not including the mutation to be tested, the father + not including the mutation to be tested, the mother + including the mutation to be tested and the mother + not including the mutation to be tested, finally, six complete sequences can be spliced, which are derived from different chromosomal strands in the ancestor and parent, respectively. Next, by first scanning all positions of the sequences of the prover and the parent containing the mutation to be tested, and by scanning the sequences of the prover and the parent containing the chr16:30097630:c > T mutation, it can be found that the base of the three at the chr16:30097660 position is different, the prover and the mother have a deletion mutation of TG > T at the chr16:30097660 position, the number of comparison results of TG > T mutation of the prover at the position is 40, the number of comparison results of TG > T mutation of the mother at the position is 34, and the father does not have the mutation at the position. Thus, according to the above-described judgment, the chr16:30097660:TG > T mutation of the precursor was inherited from the mother, further deducing that the chr16:30097630:C > T mutation in the precursor, which is on the same strand as chr16:30097660:TG > T, is inherited from the mother.
The comparison result of three families corresponding to the test at the chr16:30097630:C > T locus is shown in fig. 6, wherein the mutation locus on the left side is a target locus chr16:30097630:C > T to be tested, and the mutation locus on the right side is chr16:30097660:TG > T which can assist in judgment, and the illustrated result is consistent with the information and the result obtained in the detection step, so that the method for judging the parent heterozygous mutation source according to the comparison result of three families is reflected to be accurate.
Test 2
In another test example, the ancestral precursor in this family exhibited symptoms of ossifying myositis and bone dysplasia, and both the ancestral precursor and parent of this family were subjected to second generation sequencing for investigation of the causative agent of the disease. Based on the mutation detection of the second generation sequencing results of the forerunner and the parents, the forerunner was found to have a heterozygous mutation chr20:57478756:G > GC at the chr20:57478756 position, whereas the mutation at this position was closely related to the occurrence of the disease in the parents. Thus, in this test example, a parental source analysis was performed on the target site chr20:57478756 shown in FIG. 7 according to the method for detecting a point mutation of the parental source shown in FIG. 1. In the determination of the parental source chain in which the mutation is located, all the comparison results from three families including the target site chr20:57478756 are shown in FIG. 7, and it can be seen that the target mutation chr20:57478756:G > GC on the left side only appears in the comparison results of the former, so that it is confirmed that the mutation is new, and the source of the mutation cannot be directly determined. Since this site is a new mutation, the purpose of the source judgment is to analyze on which chromosome the mutation is inherited.
By using the method for detecting the new mutation source chain, all sequencing results of family members including target sites are respectively extracted according to the matching positions recorded in the comparison results, and the problematic comparison results are filtered according to the filtering method after extraction. And extracting corresponding reference sequences from the rest comparison results on the reference genome according to the comparison positions, carrying out double-sequence comparison, and confirming all normal and mutation sites on each sequencing read according to the sequence comparison results. And classifying, merging and counting all the comparison results according to four types of the forerunner+the mutation to be detected, the father+all the comparison results including the target site and the mother+all the comparison results including the target site, and finally splicing the complete sequences of the two forerunners and the base combinations of all the sites of each group of father and mother. Then, firstly, scanning all positions of the chromosome chain of the precursor including the mutation to be detected and the corresponding result of the parent, checking whether a certain base is definitely inherited from a certain parent, and if so, judging the source of the mutation to be detected. In the scan of the sequencing results of this example, it could be checked that there was a difference in the bases of the prover and parent at the chr20:57478807 position, the strand of the prover containing the mutation of interest did not mutate at this position, and the number of comparisons at this position where no mutation occurred was 30; from the result of the father at the site, the father has homozygous mutation of chr20:57478807:C > T at the site, and all the comparison results of the mutation at the site are 18; from the results of this locus of the mother, it was found that the heterozygous mutation of chr20:57478807:C > T occurred at this locus, all the comparison results were 11 for the mother with the mutation at this locus, and 21 for the mother without the mutation at this locus. Therefore, according to the method for judging the source of the new mutation, the wild base of the forerunner at the position chr20:57478807 can be judged to be clearly inherited from the mother, and according to the positional relationship between the wild base and the mutation chr20:57478756:G > GC to be detected of the forerunner on the same strand, the mutation chr20:57478756:G > GC of the forerunner is inferred to occur on the chromosome chain inherited from the mother.
The comparison result of three families corresponding to the test near chr20:57478756 is shown in fig. 7, wherein the insertion mutation at the left side of the diagram is a target mutation to be detected, the mutation at chr20:57478807:C > T at the right side of the diagram is a mutation site which can be used for assisting in judging the source of the target mutation, and the graphical result is consistent with the information and the result obtained in the detection step, so that the method for judging the source of the new mutation according to the comparison result of three families is reflected to be accurate.
The foregoing is a further detailed description of the present application in connection with the specific embodiments, and it is not intended that the practice of the present application be limited to such descriptions. It will be apparent to those skilled in the art to which the present application pertains that several simple deductions or substitutions may be made without departing from the spirit of the present application.
Claims (10)
1. A method of detecting a point mutation parent source, characterized by: comprises the steps of,
the comparison result obtaining step comprises the step of obtaining second-generation sequencing comparison results of ancestral three persons of a forerunner and a parent containing the position of the mutation site to be detected, namely, the result formed by comparing the original second-generation sequencing results to the matching position on the reference genome;
Genotype analysis, namely, analyzing the genotype of mutation at a mutation site to be detected, namely, homozygosity or heterozygosity, and the genotypes of parents at the site according to a second-generation sequencing comparison result;
the preliminary parent source judging step comprises the steps of carrying out preliminary parent source judging according to genotypes and the following rules: (1) The mutation site to be detected is homozygous mutation, and the parent sources are judged to be both parents; (2) The mutation site to be detected is heterozygous mutation, one of parents of the site has no mutation, and the other parent has mutation, so that the mutation site to be detected is derived from the mutant parent; (3) The mutation site to be detected is heterozygous mutation, one of parents of the site is homozygous mutation, the other parent is heterozygous mutation, and the mutation site to be detected is derived from one of homozygous mutation; (4) The mutation site to be detected is heterozygous mutation, and both parents at the site are heterozygous mutation, and the parent heterozygous mutation is marked as a parent heterozygous site; (5) Marking the mutation site to be detected as new mutation if both parents have no mutation at the corresponding site of the mutation site;
the parent heterozygous site source judging step comprises the steps of respectively extracting all comparison results containing mutation sites to be detected from comparison results of a prover and a parent, obtaining corresponding reference sequences of all comparison results according to the comparison positions of the sequences to a reference genome, and comparing the result sequences with the reference sequences in a double sequence manner; analyzing and obtaining the corresponding position relation between all bases on each comparison sequence and the reference genome sequence according to the analysis of the double-sequence comparison result, and further confirming all normal and mutation sites on each sequencing result; recording the normal and mutation sites in all the comparison results, including the bases appearing on the recording sites and the times of the base appearance, counting the bases of all the sites, and judging the parent sources according to the following method: according to comparison results of all the mutations to be detected, respectively splicing to obtain a sequence containing the mutations to be detected of each of a foreigner and a parent, traversing all recorded base positions, checking whether base definitely inherits from one of the two parents at the base positions, and judging that the source of the mutation site to be detected is the parent if the base definitely inherits from the one base exists; if the method I can not find that any base is definitely derived from a certain party of both parents, respectively counting all comparison results which contain the site but are not mutated at the site in comparison results of the foreigners and the parents, respectively splicing to obtain a sequence which does not contain the mutation to be detected, traversing all base positions in the same way, checking whether the sites have bases definitely inherited from the certain party, if the bases definitely inherit from the certain party, judging that the sequence which is not mutated at the site in the foreigners is from the certain party, and the mutation at the site is from the other party, namely the mutation site to be detected is derived from the other party;
The new mutation judging step comprises the steps of respectively extracting all comparison results containing the site to be detected from comparison results of a prover and a parent, obtaining corresponding reference sequences of all comparison results according to the comparison positions of the sequences to a reference genome, and comparing the result sequences with the reference sequences in a double sequence manner; analyzing and obtaining the corresponding position relation between all bases on each comparison sequence and the reference genome sequence according to the analysis of the double-sequence comparison result, and further confirming all normal and mutation sites on each sequencing result; recording the normal and mutation sites in all comparison results, including recording the bases appearing on the sites and the times of the base appearing, and counting the bases of all the sites; parent source judgment is carried out according to the following method: the method comprises the steps of (1) splicing to obtain a sequence containing the mutation to be detected of a prover according to all comparison results containing the mutation to be detected, wherein the sequence contains the mutation to be detected of the prover, and the base combination from two chromosome chains is obtained after the parent results are spliced, traversing all positions of the sequence spliced by the prover and the result obtained by the parent in combination, judging whether the base definitely originates from one of the two parents exists at the positions of the prover, and judging that the source of the new mutation site is the one if the base definitely inherited from the one of the two parents exists at the positions; in the method (2), if the method (1) cannot find that any base is definitely derived from one of the parents, splicing again from the comparison result of the prover to obtain a sequence of the prover, which does not contain the mutation to be detected, and then combining the sequence with the parent result obtained by splicing in the method (1), repeating the traversing steps in the method (1), judging whether any base of the prover on one chain which does not have the mutation is directly inherited from one of the parents, and if the base inherited from one of the parents is found, judging that the chain which does not have the mutation is derived from the other party, and judging that the chain which contains the mutation is derived from the other parent; and (3) if the method (1) and the method (2) cannot judge which of the parents comes from the newly mutated strand, carrying out reckoning on all bases of comparison results of the prover and the parents near the site according to the positions to obtain base genotypes of all the positions, searching the sites of heterozygous mutation of the prover and the parents, namely adjacent heterozygous sites, judging parent sources of the adjacent heterozygous sites by adopting a parent heterozygous site source judging step, and deducing parent sources of the newly mutated sites according to the parent sources of the adjacent heterozygous sites.
2. The method according to claim 1, characterized in that: in the step of obtaining the comparison result, the second-generation sequencing comparison result comprises the matching degree of the sequencing result to the position in the reference genome, the sequencing quality and the reading section and the reference genome sequence.
3. The method according to claim 1, characterized in that: in the parent heterozygous site source judging step, only more than 5 bases are reserved at each position;
in the new mutation judgment step, only the bases present at more than 5 times at each site are retained in the statistics for each sample.
4. A method according to any one of claims 1-3, characterized in that: in the new mutation judgment step, the adjacent heterozygous sites are heterozygous mutations in the base site range traversed by the method (1) or the method (2).
5. A device for detecting a point mutation parent source, characterized in that: the system comprises a comparison result acquisition module, a genotype analysis module, a parent source preliminary judgment module, a parent heterozygous site source judgment module and a new mutation judgment module;
the comparison result acquisition module is used for acquiring second-generation sequencing comparison results of the ancestral three persons of the forensics and parents containing the position of the mutation site to be detected, namely, the results formed after the original second-generation sequencing results are compared to the matching positions on the reference genome;
The genotype analysis module is used for analyzing and obtaining genotypes of mutations generated by mutation sites to be detected, namely homozygosity or heterozygosity, and genotypes of parents at the sites according to second-generation sequencing comparison results;
the parent source preliminary judgment module is used for carrying out parent source preliminary judgment according to genotypes and the following rules: (1) The mutation site to be detected is homozygous mutation, and the parent sources are judged to be both parents; (2) The mutation site to be detected is heterozygous mutation, one of parents of the site has no mutation, and the other parent has mutation, so that the mutation site to be detected is derived from the mutant parent; (3) The mutation site to be detected is heterozygous mutation, one of parents of the site is homozygous mutation, the other parent is heterozygous mutation, and the mutation site to be detected is derived from one of homozygous mutation; (4) The mutation site to be detected is heterozygous mutation, and both parents at the site are heterozygous mutation, and the parent heterozygous mutation is marked as a parent heterozygous site; (5) Marking the mutation site to be detected as new mutation if both parents have no mutation at the corresponding site of the mutation site;
the parent heterozygous site source judging module is used for respectively extracting all comparison results containing the mutation site to be detected from comparison results of a precursor and a parent, comparing sequences of all the comparison results to the position of a reference genome according to the comparison results to obtain corresponding reference sequences, and comparing the result sequences with the reference sequences in a double sequence manner; analyzing and obtaining the corresponding position relation between all bases on each comparison sequence and the reference genome sequence according to the analysis of the double-sequence comparison result, and further confirming all normal and mutation sites on each sequencing result; recording the normal and mutation sites in all the comparison results, including the bases appearing on the recording sites and the times of the base appearance, counting the bases of all the sites, and judging the parent sources according to the following method: according to comparison results of all the mutations to be detected, respectively splicing to obtain a sequence containing the mutations to be detected of each of a foreigner and a parent, traversing all recorded base positions, checking whether base definitely inherits from one of the two parents at the base positions, and judging that the source of the mutation site to be detected is the parent if the base definitely inherits from the one base exists; if the method I can not find that any base is definitely derived from a certain party of both parents, respectively counting all comparison results which contain the site but are not mutated at the site in comparison results of the foreigners and the parents, respectively splicing to obtain a sequence which does not contain the mutation to be detected, traversing all base positions in the same way, checking whether the sites have bases definitely inherited from the certain party, if the bases definitely inherit from the certain party, judging that the sequence which is not mutated at the site in the foreigners is from the certain party, and the mutation at the site is from the other party, namely the mutation site to be detected is derived from the other party;
The new mutation judging module is used for respectively extracting all comparison results containing the site to be detected from comparison results of a prover and a parent, obtaining corresponding reference sequences of all comparison results according to the positions of the comparison results to a reference genome, and comparing the result sequences with the reference sequences in a double sequence manner; analyzing and obtaining the corresponding position relation between all bases on each comparison sequence and the reference genome sequence according to the analysis of the double-sequence comparison result, and further confirming all normal and mutation sites on each sequencing result; recording the normal and mutation sites in all the comparison results, including the bases appearing on the recording sites and the times of the base appearance, counting the bases of all the sites, and judging the parent sources according to the following method: the method comprises the steps of (1) splicing to obtain a sequence containing the mutation to be detected of a prover according to all comparison results containing the mutation to be detected, wherein the sequence contains the mutation to be detected of the prover, and the base combination from two chromosome chains is obtained by splicing the parent results, traversing all positions of the sequence spliced by the prover and the result obtained by splicing the parent, judging whether the base definitely originates from one of the two parents exists at the positions of the prover, and judging that the source of the new mutation site is the one if the base definitely inherits from the one of the two parents exists at the positions; in the method (2), if the method (1) cannot find that any base is definitely derived from one of the parents, splicing again from the comparison result of the prover to obtain a sequence of the prover, which does not contain the mutation to be detected, and then combining the sequence with the parent result obtained by splicing in the method (1), repeating the traversing steps in the method (1), judging whether any base of the prover on one chain which does not have the mutation is directly inherited from one of the parents, and if the base inherited from one of the parents is found, judging that the chain which does not have the mutation is derived from the other party, and judging that the chain which contains the mutation is derived from the other parent; and (3) if the method (1) and the method (2) cannot judge which of the parents comes from the newly mutated strand, carrying out reckoning on all bases of comparison results of the prover and the parents near the site according to the positions to obtain base genotypes of all the positions, searching the sites of heterozygous mutation of the prover and the parents, namely adjacent heterozygous sites, judging parent sources of the adjacent heterozygous sites by adopting the parent heterozygous site source judging module, and deducing parent sources of the newly mutated sites according to the parent sources of the adjacent heterozygous sites.
6. The apparatus according to claim 5, wherein: in the comparison result acquisition module, the second-generation sequencing comparison result comprises the matching degree of the sequencing result to the position in the reference genome, the sequencing quality and the reading section and the reference genome sequence.
7. The apparatus according to claim 5, wherein: in the parent heterozygous site source judging module, each position only retains more than 5 bases;
in the new mutation judgment module, only bases which appear more than 5 times at each site are reserved in statistics of each sample.
8. The apparatus according to any one of claims 5-7, wherein: in the new mutation judgment module, the adjacent heterozygous sites are heterozygous mutations in the base site range traversed by the method (1) or the method (2).
9. A device for detecting a point mutation parent source, characterized in that: the apparatus includes a memory and a processor;
the memory comprises a memory for storing a program;
the processor comprising means for implementing the parent source of the point mutation of any one of claims 1-4 by executing the program stored in the memory.
10. A computer-readable storage medium, characterized by: the storage medium having stored therein a program executable by a processor to perform the method of detecting a parent source of a point mutation of any of claims 1-4.
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