Detection and analysis method of ultra-low frequency mutation sites based on duplex-seq

Abstract

A duplex-seq-based ultra-low frequency mutation site detection and analysis method disclosed in the present invention comprises the following steps: 1) evaluating the quality of original sequencing data, reducing data noise, and providing effective data for subsequent analysis; 2) random barcode Extract the title line of each sequence of the sequence file, which is convenient for subsequent quick retrieval of barcode and creation of consistent sequences; 3) Create consistent sequences based on family barcode and duplex barcode, and exclude mutations introduced during the library construction process or PCR process ; 4) Construct a double-stranded consensus sequence according to duplex-tag, and further exclude asymmetric mutation sites in the sequence; 5) Perform local quality correction on the compared data, and perform low-frequency mutation site detection; Three-level annotation of gene structure, function, and clinical phenotype; 6) Statistics of SSCS, DCS sequence numbers, comparison results, and variation site information, and output visual charts.

Description

technical field [0001] The invention belongs to a biological information data processing method, in particular to a duplex-seq-based ultra-low frequency mutation site detection and analysis method, which is mainly used in the field of second-generation high-throughput sequencing, based on duplex-seq whole exome sequencing , to detect and analyze the ultra-low frequency mutation sites of ctDNA. Background technique [0002] The development of next-generation sequencing technology is in full swing, and it is profoundly changing the research of traditional genetics with overwhelming power, and thus giving birth to the germination of precision medicine. Compared with traditional experimental techniques, this technique can detect thousands of genetic mutations at one time. However, the fly in the ointment is that the next generation sequencing technology still has a relatively high error rate (0.1-1%). For the detection of high-frequency genetic mutations, this error is accepta...

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Problems solved by technology

[0005] 1. Data quality control: In the existing Duplex-seq data analysis process, there is no systematic analysis of the previous data quality, such as data repetition rate, UMI type, quantity, ratio, R1R2 balance, etc.

[0006] 2. Differential analysis of UMI: The comprehensive and independent analysis of single-strand-specific UMI and double-strand complementary UMI has not been reported in the existing Duplex-seq data analysis methods

[0007] 3. Variant site annotation process: The mutation sites copied out based on Duplex-seq data belong to low-frequency mutation sites. Therefore, the existing methods can be optimized for the parameters related to the detection of variant sites. not systematic enough

[0008] 4. Readability of results: In the existing Duplex-seq data analysis methods, there are only some simple chart files and text reports in the results, and a lot of data information is not effectively presented

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