Next generation sequencing-based point mutation detection filtering method and apparatus, and storage medium
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A next-generation sequencing and point mutation technology, applied in genomics, sequence analysis, proteomics, etc., can solve the problems of low detection specificity, inability to detect samples and sites at the same time, long running time, etc.
Active Publication Date: 2018-04-20
深圳裕策生物科技有限公司
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Problems solved by technology
The PCR method has the characteristics of high sensitivity, and the technology is mature, but each pair of primers can only detect one mutation, cannot detect too many samples and sites at the same time, and the throughput is low
The cost of Sanger sequencing is low, but the amount of sample required is large, and the detection sensitivity for low-frequency mutations is low
Next-generation sequencing has the characteristics of hig
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Embodiment 1
[0112] In this embodiment, the samples used are standard products purchased from Horizon, and the samples to be tested include 3 positive standard products Q1, Q3 and Q5, and the theoretical VAFs corresponding to the positive sites are 1%, 3% and 5% respectively; There is also a negative control sample Q0. The specific steps of paired sample detection in this embodiment are as follows:
[0113] (1) Using the BAM files of the positive standard products Q1, Q3, Q5 and the control sample Q0 respectively, obtain the set of candidate somatic point mutation sites of the three samples to be tested.
[0114] (2) Obtain the unfiltered point mutation results of the three samples to be tested through the primary filtering step, and then count the number of mutation supports and VAF at the corresponding positions in the control sample.
[0115] (3) Statistically compare detailed information at and around the site obtained after the primary filtering step in the three samples to be tested...
Embodiment 2
[0129] In this embodiment, the sample to be tested is a quality-assessed point mutation-positive sample, including 3 positive point mutation sites, and the VAF is 1%-20%. The specific steps of single-sample detection in this embodiment are as follows:
[0130] (1) Using the BAM file of the sample to be tested, a set of candidate point mutation sites is obtained.
[0131] (2) Obtain the unfiltered point mutation result of the sample to be tested through the primary filtering step.
[0132] (3) Statistically compare detailed information at and around the site obtained after the primary filtering step in the sample to be tested.
[0133] (4) Through the advanced filtering module, the unfiltered point mutation detection results in the samples to be tested are finally obtained.
[0134] The sensitivity of the final detection result of this embodiment to the 3 positive sites is 100%.
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Abstract
The invention discloses a next generation sequencing-based point mutation detection filtering method and apparatus, and a storage medium. The method comprises the steps of performing comparison with files of a reference genome by utilizing a to-be-detected sample, and extracting candidate point mutation site sets with variant allele frequencies exceeding a set threshold; preliminarily calculatingsupport numbers of mutant bases and reference bases of candidate point mutation sites, and filtering results with the mutant support numbers lower than the set threshold and/or the variant allele frequencies lower than the set threshold; performing detailed statistics on the candidate point mutation sites and surrounding comparison information, wherein the information comprises at least one of thefollowing information: the support numbers of the mutant bases and the reference bases of the candidate point mutation sites, base and comparison quality, coverage depth, surrounding non reference base and insertion/deletion statuses, and surrounding read quality; and according to the statistical information, filtering the results which do not meet set requirements to obtain a point mutation detection result. While the resource demands and the detection speed are optimized, the sensitivity and specificity of point mutation detection are improved.
Description
technical field [0001] The invention relates to the technical field of mutation detection, in particular to a point mutation detection and filtering method, device and storage medium based on next-generation sequencing. Background technique [0002] At present, common methods for detecting gene point mutations include PCR method, Sanger sequencing method (first-generation sequencing) and next-generation sequencing. The PCR method has the characteristics of high sensitivity, and the technology is mature, but each pair of primers can only detect one mutation, cannot detect too many samples and sites at the same time, and the throughput is low. The cost of Sanger sequencing is low, but the amount of sample required is large, and the detection sensitivity for low-frequency mutations is low. Next-generation sequencing has the characteristics of high throughput, and the cost of sequencing is also decreasing year by year, but the current methods and tools commonly used to detect p...
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