Primer system for multiple amplicon sequencing, application thereof and building method of sequencing library

Abstract

The invention discloses a primer system for multiple amplicon sequencing, application thereof and a building method of a sequencing library. The primer system comprises a first-round reverse specificprimer group and a second-round forward specific primer group, the first-round reverse specific primer group comprises multiple different first-round reverse specific primers, and 5' end, 4' end and 3' end of each reverse specific primer are in reverse joint sequence, barcode sequence and reverse specific sequence. The molecular barcode sequence provides a good method for tracking mispairing, mispairing of polymerase in the process of PCR can be differentiated from sequence variation in original molecules by utilizing downstream analysis, and mutation detection accuracy of 1% or lower is improved by removing low-level false positive rate. Target quantification can be realized better by calculating unique molecular barcode sequence number in reading rather than calculating number of total reading as the total reading is more likely to be misread by heterogeneous amplification.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a primer system for multiple amplicon sequencing, its application and a method for constructing a sequencing library. Background technique [0002] High-throughput sequencing (also known as next-generation sequencing, NGS) technology has gradually become a widely used technology in many aspects of discovery and translational research. At present, it is mainly used in the variation analysis of genomic DNA and the expression analysis of RNA. These analyzes can range from the entire genome and transcriptome to targeted sequencing of specific regions or combinations of multiple genes. Targeted sequencing is especially beneficial for achieving high coverage of regions of interest while controlling sequencing cost and data interpretation complexity. High sequencing coverage is especially important for low-frequency mutations such as cancer, for example, for detecting single nucleotide...

AI Technical Summary

Problems solved by technology

These problems are exacerbated when more PCR cycles are required to process low-input DNA or poor-quality DNA

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