Method for correcting base variation detection results and application thereof

Abstract

The application discloses a method for correcting base variation detection results and application thereof. In view of the problem that existing variation detection methods cannot combine variations of triple codons coding the same amino acid and amino acid changes caused by the variations, the application develops a method for correcting the detection results of variation detection software, corrects amino acid changes caused by triple codon mutation according to upstream sequencing sequences of variation sites, and obtains real variations of bases and amino acids.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological information, and relates to a method for correcting base variation detection results and application thereof. BACKGROUND

[0002] The progress of targeted therapy is closely related to the progress of sequencing technology in the past two decades, including the revolutionary development of large-scale parallel next generation sequencing (NGS). The discovery of common and rare genetic abnormalities has initiated targeted therapy research for the resulting mutant proteins and explored downstream abnormal molecular signaling pathways that can be used for treatment. In addition, NGS has been shown to be critical to the clinical application of targeted therapy, and mutation evaluation by sequence oligonucleotide capture, amplification and NGS has become a standard diagnostic tool for many cancers, and these tests are used to identify operable genetic mutations, which are then used to select appropriate targeted therapies, predict or serve as biomarkers for other clinical endpoints.

[0003] Single base mutations and short insertion-deletion mutations as targetable mutations have changed the treatment strategies of many cancers such as non-small cell lung cancer and colorectal cancer. The accurate detection of these mutations also greatly influences the development of treatment strategies. For multiple single base mutations such as EGFR L858R and EGFR T790M, various targeted drugs have been approved for the treatment of non-small cell lung cancer patients with corresponding mutations at home and abroad.

[0004] Existing mutation detection software usually identifies mutations at a single base position, while one or more mutations in three consecutive bases that encode the same codon should be considered together, for example, a sample VarScan detection result is chr2:25398284C>A:p.G12V and chr2:25398285C>A:p.G12C, which occur simultaneously, if viewed as a single base mutation, it is 2 mutations, in fact, the bases at the two positions are two bases in the same triple codon, and the corrected variation is chr2:25398284_25398285CC>AA:p.G12F. Therefore, the detection results of the variation detection software should be considered comprehensively and corrected to obtain the real variation of the bases and amino acids.

[0005] In summary, the development of a correction method that can accurately analyze the real situation of the variation is of great significance to the field of targeted therapy. SUMMARY

[0006] In view of the deficiencies of the prior art and actual needs, the application provides a method for correcting base variation detection results and application thereof, which corrects the detection results of mutation detection software.

[0007] To achieve the above object, the present application adopts the following technical solutions:

[0008] In a first aspect, the present application provides a method for correcting base variation detection results, comprising the following steps:

[0009] (1) inputting annotated variation detection results and aligned bam files;

[0010] (2) obtaining the cDNA position corresponding to the variation position according to the annotation results;

[0011] (3) determining the gene where the mutation occurs according to the annotation results, and obtaining the gene direction from a database;

[0012] (4) determining the absolute position of the triple codon corresponding to the variation position by using the remainder method:

[0013] (5) according to the starting position of the triple codon, grabbing the reference sequence of the triple codon from the reference genome;

[0014] If the gene is a positive strand gene, the triple codon reference sequence remains unchanged; if it is a negative strand gene, the triple codon reference sequence is sorted in reverse, and then converted according to the base complementary pairing principle;

[0015] (6) determining the corresponding amino acid according to the triple codon reference sequence;

[0016] (7) grabbing all sequencing sequences covering the triple codon from the bam file;

[0017] (8) according to the cigar value and the starting position of the sequencing sequence, the sum of the base numbers corresponding to the starting position and the identification characters "M / I" to obtain the termination position of the sequencing sequence;

[0018] (9) including the sequencing sequences covering the three bases of the triple codon into subsequent analysis;

[0019] (10) correcting the sequencing sequence of the mutation according to the cigar value: traversing the cigar value, the identification character is "M", then retaining the base sequence corresponding to the identification character; the identification character is "I", then retaining the base sequence corresponding to the identification character; the identification character is "D / S / H", then ignoring; concatenating the retained base sequences;

[0020] (11) according to the starting position of the corrected sequencing sequence and the starting position of the triple codon, determining the three base sequences located at the position of the triple codon on each sequencing sequence;

[0021] (12) According to step (5), if it is a positive strand gene, the three-base sequence of the triple code is kept unchanged, and if it is a negative strand gene, the three bases of the triple code are sorted in reverse order;

[0022] (13) The amino acid corresponding to the triple code sequence determined in step (12) is compared with the amino acid corresponding to the reference sequence of the triple code determined in step (5), if the two are different, it is considered that the triple code has a mutation, and the starting and ending positions, the reference sequence, the mutation sequence, the reference amino acid and the mutation amino acid information are recorded;

[0023] (14) The mutated triple code base sequence is compared with the reference sequence, the first base position and the last base position of the mutation are recorded, and the mutation sequence and the reference sequence corresponding to the mutation sequence are retained, that is, the final mutant chromosome, the mutation position, the reference sequence, the mutation sequence, the reference amino acid and the mutation amino acid.

[0024] In view of the problems that the existing variation detection method cannot combine the variation of the triple code coding the same amino acid and the amino acid change caused thereby, the data correction method designed by the application corrects the amino acid change caused by the mutation of the triple code according to the upstream sequencing sequence of the variation site, realizes accurate detection of the variation, and avoids false positive and false negative results.

[0025] In the application, the bam file refers to a result file generated after the sequencing data is aligned by using alignment software; the cigar is a combination of numbers and letters, and records the detailed alignment of the sequencing sequence to the reference sequence.

[0026] Preferably, the database in step (3) includes any one of refGene database, GeneBank or Ensemble.

[0027] Preferably, step (4) specifically includes:

[0028] The cDNA position is divided by 3 and the remainder is obtained;

[0029] If the remainder is 0, let the remainder = 3;

[0030] If the gene is a positive strand gene, the starting position of the triple code is variation position-remainder+1, and the ending position is variation position-remainder+3;

[0031] If the gene is a negative strand gene, the starting position of the triple code is variation position+remainder-3, and the ending position is variation position+remainder-1.

[0032] In a second aspect, the application provides an application of the method for correcting the base variation detection result in the gene variation detection.

[0033] In a third aspect, the present application provides a method for detecting base variation, comprising:

[0034] sequencing the sample to be detected, performing variation analysis on the sequencing data to obtain a base variation detection result, and correcting the detection result using the method for correcting base variation detection result according to the first aspect.

[0035] Preferably, the variation analysis comprises using mutation analysis software for analysis.

[0036] Preferably, the mutation analysis software comprises any one of VarScan, Mutect2, GATK, Lofreq, Strelka or SomaticSniper.

[0037] It can be understood that the method of the present application can correct the detection result of the existing mutation analysis software, such as solving the problem that VarScan cannot combine the triple codon variation encoding the same amino acid and the amino acid change caused thereby, and the problem that Mutect2 cannot process the variation of the first and third bases, and has important application value in the fields of diagnosis and basic behavior research of gene variation.

[0038] In a fourth aspect, the present application provides a device for correcting base variation detection result, which is used to perform a method comprising the following steps:

[0039] (1) inputting the annotated variation detection result and the aligned bam file;

[0040] (2) obtaining the cDNA position corresponding to the variation position according to the annotation result;

[0041] (3) determining the gene where the mutation occurs according to the annotation result, and obtaining the gene direction from the database;

[0042] (4) determining the absolute position of the triple codon corresponding to the variation position using the remainder method:

[0043] (5) according to the starting position of the triple codon, grabbing the reference sequence of the triple codon from the reference genome;

[0044] If the gene is a positive strand gene, the triple codon reference sequence remains unchanged; if it is a negative strand gene, the triple codon reference sequence is sorted in reverse, and then converted according to the base complementary pairing principle;

[0045] (6) determining the corresponding amino acid according to the triple codon reference sequence;

[0046] (7) grabbing all sequencing sequences covering the triple codon from the bam file;

[0047] (8) According to the cigar value and the starting position of the sequencing sequence, the starting position plus the sum of the identification characters "M / I" and the corresponding base number to obtain the ending position of the sequencing sequence;

[0048] (9) The sequencing sequence covering three bases of the triple codon is incorporated into subsequent analysis;

[0049] (10) According to the cigar value, the sequencing sequence of the mutation is corrected: the cigar value is traversed, the identification character is "M", the base sequence corresponding to the identification character is retained; the identification character is "I", the base sequence corresponding to the identification character is retained; the identification character is "D / S / H", it is ignored; the retained base sequence is concatenated;

[0050] (11) According to the starting position of the corrected sequencing sequence and the starting position of the triple codon, the three base sequences located at the position of the triple codon on each sequencing sequence are determined;

[0051] (12) According to step (5), if it is a positive strand gene, the three base sequences of the triple codon remain unchanged, and if it is a negative strand gene, the three base sequences of the triple codon are sorted in reverse order;

[0052] (13) The amino acid corresponding to the triple codon sequence determined in step (12) is compared with the amino acid corresponding to the reference sequence of the triple codon determined in step (5). If they are different, it is considered that the triple codon has a mutation, and the starting and ending positions, the reference sequence, the mutant sequence, the reference amino acid and the mutant amino acid information are recorded;

[0053] (14) The mutated triple codon base sequence is compared with the reference sequence, the first base position and the last base position where the mutation occurs are recorded, and the mutant sequence and the reference sequence corresponding to the mutant sequence are retained, which is the final mutant chromosome, mutation position, reference sequence, mutant sequence, reference amino acid and mutant amino acid.

[0054] Preferably, the database of step (3) includes any one of refGene database, GeneBank or Ensemble.

[0055] Preferably, step (4) specifically includes:

[0056] The cDNA position is divided by 3 and the remainder is taken;

[0057] If the remainder is 0, let the remainder = 3;

[0058] If the gene is a positive strand gene, the starting position of the triple codon is the variation position-remainder+1, and the ending position is the variation position-remainder+3;

[0059] If the gene is a negative strand gene, the start position of the triplet codon is the variation position + remainder-3, and the end position is the variation position + remainder-1.

[0060] In a fifth aspect, the present application provides a computer readable storage medium storing a computer program, which causes a computer to establish and / or run the steps of the method for manufacturing simulated variation data according to the first aspect.

[0061] Compared with the prior art, the present application has the following beneficial effects:

[0062] In view of the problem that the existing variation detection method cannot combine the variation of the triplet codon coding the same amino acid and the amino acid change caused thereby, the present application designs a brand-new data correction method, corrects the amino acid change caused by the mutation of the triplet codon according to the upstream sequencing sequence of the variation site, realizes accurate detection of the variation, and avoids false positive and false negative results. DETAILED DESCRIPTION

[0063] In order to further illustrate the technical means adopted by the present application and its effects, the present application will be further described below in conjunction with examples. It can be understood that the specific embodiments described herein are only used to explain the present application, and not to limit the present application.

[0064] If the specific technology or condition is not specified in the examples, the technology or condition described in the literature in the art or according to the product manual is used. If the reagent or instrument is not specified by the manufacturer, it is a conventional product that can be purchased through a regular channel.

[0065] Example 1

[0066] In this example, the detection results of each variation detection software annotated by ANNOVAR (as shown in Table 1) are used as an example to verify the correction method of the present application.

[0067] Table 1

[0068]

[0069]

[0070]

[0071] Specific methods include:

[0072] 1. Input two variation sites and their variation information and the obtained BAM file after alignment;

[0073] 2. The cDNAs corresponding to the two variations are 35 and 34, respectively;

[0074] 3. The two mutations are located on the KRAS gene, which is a negative strand gene. According to the remainder method (cDNA position divided by 3, if the remainder is 0, let the remainder = 3; if the gene is a positive strand gene, the start position of the triple codon is the mutation position - remainder + 1, and the end position is the mutation position - remainder + 3; if the gene is a negative strand gene, the start position of the triple codon is the mutation position + remainder - 3, and the end position is the mutation position + remainder - 1.), the absolute positions of the triple codons corresponding to the two mutations are both chr12:25398283-25398285;

[0075] 4. The sequence of chr12:25398283-25398285 on the human reference genome Hg19 is ACC, and since KRAS is a negative strand gene, the base sequence is sorted in reverse order, resulting in GGT, which corresponds to the amino acid glycine (Gly, G);

[0076] 5. All sequencing sequences covering chr12:25398283-25398285 are extracted from the bam file;

[0077] 6. According to the cigar value and the start position of the sequencing sequence, the end position of the sequencing sequence is determined, and only the sequencing sequences covering both chr12:25398283 and chr12:25398285 are included for subsequent analysis;

[0078] 7. According to the cigar value, the sequencing sequence of the mutation is corrected: the cigar value is traversed, and if the identification character is "M", the base sequence corresponding to the identification character is retained; if the identification character is "I", the base sequence corresponding to the identification character is retained; if the identification character is "D / S / H", it is ignored; the retained base sequence is concatenated;

[0079] 8. According to the start position of the corrected sequencing sequence and the start position of the triple codon, the three base sequences located at the position of the triple codon on each sequencing sequence are determined;

[0080] 9. The triple codon base sequence and the gene direction determine the corresponding amino acid, and if the amino acid at this position is not glutamic acid, the mutation information at this position is retained;

[0081] 10. The base sequence of the triple codon corresponding to the amino acid that is not glutamic acid is compared with the reference sequence, and the first and last base positions of the mutation are retained, as well as the mutant base sequence and the reference sequence, as shown in Table 2;

[0082] Table 2

[0083]

[0084]

[0085] 11. Filter out mutations with sequencing depth less than 1000, mutant base support sequence number less than 3, and mutation frequency less than 0.002, and the results are shown in Table 3;

[0086] Table 3

[0087]

[0088] 12. The corrected results are consistent with the results of Mutect2.

[0089] It is shown that the correction method of the present application corrects the detection results of the existing variant detection software according to the amino acid changes caused by the triple codon mutation of the upstream sequencing sequence of the variant site.

[0090] Example 2

[0091] Mutect2 can combine to detect the variation of adjacent bases in the triple codon, and cannot process the variation of the first and third bases. The present application uses the correction method to correct the results, and the specific implementation process is as follows:

[0092] 1. Data simulation is performed for the mutation information in Table 4

[0093] Table 4

[0094]

[0095] 2. The variant detection results of Mutect2 annotated by Annovar are shown in Table 5;

[0096] Table 5

[0097]

[0098]

[0099] 3. The above results are corrected, and the results are shown in Table 6 according to Example 1.

[0100] Table 6

[0101]

[0102] The results show that the method for correcting the base variation detection results of the present application can correct the variation results to obtain intuitive and correct correction results.

[0103] In summary, the present application develops a method for correcting the detection results of the variant detection software, and corrects the amino acid changes caused by the triple codon mutation according to the upstream sequencing sequence of the variant site to obtain the true variation of the base and amino acid.

[0104] Applicants declare that the detailed methods of the present application are illustrated by the above examples, but the present application is not limited to the above detailed methods, i.e. it is not meant that the present application must rely on the above detailed methods to be implemented. It should be understood by those skilled in the art that any improvement on the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific methods, etc. all fall within the protection scope and disclosure scope of the present application.

Claims

1. A method for correcting base variation detection results, characterized in that, The method includes the following steps: (1) Input the annotated mutation detection results and the aligned BAM file; (2) Obtain the cDNA location corresponding to the mutation location based on the annotation results; (3) Determine the gene in which the mutation occurred based on the annotation results, and obtain the gene direction from the database; (4) Use the remainder method to determine the absolute position of the triplet codon corresponding to the mutation position: (5) Extract the reference sequence of the triplet codon from the reference genome based on the start position of the triplet codon; If the gene is a positive-strand gene, the triplet codon reference sequence remains unchanged; if it is a negative-strand gene, the triplet codon reference sequence is reversed and then converted according to the base pairing principle. (6) Determine the corresponding amino acid based on the reference sequence of the triplet codon; (7) Extract all sequencing sequences covering the triplet codon from the bam file; (8) Based on the cigar value and the start position of the sequencing sequence, add the sum of the bases corresponding to the identifier "M / I" to the start position to obtain the end position of the sequencing sequence; (9) Sequencing sequences that simultaneously cover all three bases of a triplet codon were included in subsequent analysis; (10) Correct the sequencing sequence of the mutation according to the cigar value: traverse the cigar value, if the identifier is "M", retain the base sequence corresponding to the identifier; if the identifier is "I", retain the base sequence corresponding to the identifier; if the identifier is "D / S / H", ignore it; concatenate the retained base sequences. (11) Based on the start position of the corrected sequencing sequence and the start position of the triple codon, determine the three base sequences located at the triple codon position on each sequencing sequence; (12) According to step (5), if it is a positive strand gene, the three base sequences of the triplet codon remain unchanged; if it is a negative strand gene, the three bases of the triplet codon are reversed. (13) Compare the amino acid corresponding to the triple codon sequence determined in step (12) with the amino acid corresponding to the reference sequence of the triple codon determined in step (5). If they are different, the triple codon is considered to have mutated. Record its start and stop positions, reference sequence, mutated sequence, reference amino acid and mutated amino acid information. (14) Compare the mutated triplet codon base sequence with the reference sequence, record the position of the first and last bases where the mutation occurred, and retain the mutated sequence and the reference sequence corresponding to the mutated sequence. This is the final mutated chromosome, mutated position, reference sequence, mutated sequence, reference amino acid and mutated amino acid.

2. The method for correcting base variation detection results according to claim 1, characterized in that, The database mentioned in step (3) includes any one of the following: refGene database, GeneBank, or Ensemble.

3. The method for correcting base variation detection results according to claim 1 or 2, characterized in that, Step (4) specifically includes: The remainder when the cDNA location is divided by 3; If the remainder is 0, let the remainder = 3; If the gene is a positive strand gene, the start position of the triplet codon is the mutation position - remainder + 1, and the end position is the mutation position - remainder + 3; If the gene is a negative-strand gene, the start position of the triplet codon is the mutation position + remainder - 3, and the end position is the mutation position + remainder - 1.

4. The application of the method for correcting base variation detection results as described in any one of claims 1-3 in gene variation detection.

5. A method for detecting base variation, characterized in that, The method includes: The sample to be tested is sequenced, and the sequencing data is subjected to variation analysis to obtain the base variation detection results. The detection results are then corrected using the method for correcting the base variation detection results as described in any one of claims 1-3.

6. The base variation detection method according to claim 5, characterized in that, The variation analysis includes analysis using mutation analysis software.

7. A device for correcting base variation detection results, characterized in that, The device is used to perform the following steps: (1) Input the annotated mutation detection results and the aligned BAM file; (2) Obtain the cDNA location corresponding to the mutation location based on the annotation results; (3) Determine the gene in which the mutation occurred based on the annotation results, and obtain the gene direction from the database; (4) Use the remainder method to determine the absolute position of the triplet codon corresponding to the mutation position: (5) Extract the reference sequence of the triplet codon from the reference genome based on the start position of the triplet codon; If the gene is a positive-strand gene, the triplet codon reference sequence remains unchanged; if it is a negative-strand gene, the triplet codon reference sequence is reversed and then converted according to the base pairing principle. (6) Determine the corresponding amino acid based on the reference sequence of the triplet codon; (7) Extract all sequencing sequences covering the triplet codon from the bam file; (8) Based on the cigar value and the start position of the sequencing sequence, add the identifier "M / I" and the sum of its corresponding bases to the start position to obtain the end position of the sequencing sequence; (9) Sequencing sequences that simultaneously cover all three bases of a triplet codon were included in subsequent analysis; (10) Correct the sequencing sequence of the mutation according to the cigar value: traverse the cigar value, if the identifier is "M", retain the base sequence corresponding to the identifier; if the identifier is "I", retain the base sequence corresponding to the identifier; if the identifier is "D / S / H", ignore it; concatenate the retained base sequences. (11) Based on the start position of the corrected sequencing sequence and the start position of the triple codon, determine the three base sequences located at the triple codon position on each sequencing sequence; (12) According to step (5), if it is a positive strand gene, the three base sequences of the triplet codon remain unchanged; if it is a negative strand gene, the three bases of the triplet codon are reversed. (13) Compare the amino acid corresponding to the triple codon sequence determined in step (12) with the amino acid corresponding to the reference sequence of the triple codon determined in step (5). If they are different, the triple codon is considered to have mutated. Record its start and stop positions, reference sequence, mutated sequence, reference amino acid and mutated amino acid information. (14) Compare the mutated triplet codon base sequence with the reference sequence, record the position of the first and last bases where the mutation occurred, and retain the mutated sequence and the reference sequence corresponding to the mutated sequence. This is the final mutated chromosome, mutated position, reference sequence, mutated sequence, reference amino acid and mutated amino acid.

8. The apparatus for correcting base variation detection results according to claim 7, characterized in that, The database mentioned in step (3) includes any one of the following: refGene database, GeneBank, or Ensemble.

9. The apparatus for correcting base variation detection results according to claim 7 or 8, characterized in that, Step (4) specifically includes: The remainder when the cDNA location is divided by 3; If the remainder is 0, let the remainder = 3; If the gene is a positive strand gene, the start position of the triplet codon is the mutation position - remainder + 1, and the end position is the mutation position - remainder + 3; If the gene is a negative-strand gene, the start position of the triplet codon is the mutation position + remainder - 3, and the end position is the mutation position + remainder - 1.

10. A computer-readable storage medium storing a computer program, characterized in that, The computer program enables the computer to establish and / or run the steps of the method for correcting base variation detection results as described in any one of claims 1-3.

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