Next-generation sequencing library building kit and method for improving library conversion rate

Abstract

The invention relates to a method and a kit in the technical field of biology, and concretely relates to a next-generation sequencing library building kit and method for improving the library conversion rate. The kit and the method are particularly suitable for next-generation sequencing application of trace samples. The kit comprises: enzyme mixed solutions which comprises a first enzyme mixed solution, a second enzyme solution and a third enzyme mixed solution, wherein the first enzyme mixed solution is formed by mixing T4 polynucleotide kinase, T4 DNA polymerase, a Klenow fragment and Taq enzyme according to a ratio of 3: 1: 2: 1, the second enzyme solution is T4 DNA ligase, and the third enzyme mixed solution is a mixed solution of 2X QuarTaq HiFi HotStart; buffer solutions which comprise a first buffer solution corresponding to the first enzyme mixed solution and a second buffer solution corresponding to the second enzyme mixed solution, wherein the second buffer solution is formed by mixing Tris-HCl with the concentration of 0.1-0.6 M, MgCl2 with the concentration of 0.05-0.1 M, DTT with the concentration of 0.01-0.05 M, ATP with the concentration of 1-10 mM, PEG6000 with the concentration of 30%-50% and 1, 2 propylene glycol with the concentration of 10-20%; and a linker, an index sequence and a universal primer.

Description

technical field [0001] The present invention relates to methods and kits in the field of biotechnology, in particular, to a next-generation sequencing library construction kit and method for improving library conversion rates. Background technique [0002] High-throughput sequencing technology, called Next Generation Sequencing (NGS) in some literatures, is an epoch-making revolutionary change to traditional sequencing (Sanger sequencing). Thousands of DNA molecular sequences were determined. [0003] In recent years, more and more genomes including human, Arabidopsis and rice have been sequenced. Whole genome sequencing provides a solid platform for cloning of important genes, phylogenetic evolution of genes and genomics research. However, there are still some rare species with less genomic DNA (less than 50ng) due to the difficulty in obtaining samples or the low concentration of genomes obtained from small individuals. [0004] Because ctDNA carries all the mutation inf...

AI Technical Summary

Problems solved by technology

For trace amounts of DNA samples (<50ng), the conversion efficiency of library construction kits commonly used at home and abroad is relatively low. For example, Roche KAPA Biosystems’ KAPA Hyper series of library construction has a good reputation in the industry and is also the most widely used. The conversion rate of the library in the official manual of the kit is 5-40%, which is not enough for many application scenarios

Therefore, there is still a need to improve the existing technology to improve the low conversion rate of the existing micro-initial sample library.

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