Primer system for multiplex amplicon sequencing, application thereof and method for constructing sequencing library

Abstract

The invention discloses a primer system for multiple amplicon sequencing, its application and a method for constructing a sequencing library. The primer system includes a first-round reverse-specific primer set and a second-round forward-specific primer set. Wherein the first-round reverse-specific primer set includes a plurality of different first-round reverse-specific primers, and each of the reverse-specific primers is a reverse linker sequence and a barcode sequence from the 5' end to the 3' end respectively. and reverse specific sequences. Molecular barcode sequences provide an excellent way to track mismatches. Using downstream analysis, mismatches generated by the polymerase during PCR can be distinguished from sequence variations that occurred in the original molecule. Finally, by removing low-level spurious Positive to improve detection accuracy for mutations of 1% or less. Target quantification is also better achieved by counting the number of unique molecular barcode sequences in a read, rather than total reads, which are more likely to be misread by heterogeneously amplified ones.

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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