Amplification sublibrary and construction method thereof

Abstract

The invention provides an amplification sublibrary and a construction method thereof. The construction method comprises the following steps: S1, performing PCR amplification on target fragments of multiple samples to obtain a plurality of amplification products; S2, performing balanced mixing on the amplification products to obtain a mixed product; S3, sequentially performing fragmentation and terminal repairing on the mixed product, and adding A at a terminal 3' of the mixed product to obtain an A-repaired product; S4, jointing the A-repaired product by using a joint of PCR-free to obtain the amplification sublibrary. Through balanced mixing of the amplification products and through joint connection by using the optimized PCR-free, by the construction method, preference generated by PCR as well as chimeras in environmental samples is avoided, so that sequencing data between different samples have good consistency, subsequent clustering analysis and technical repeatability are more reasonable and more accurate, and can more really reflect evolutional relationship between the environmental samples.

Description

technical field [0001] The present invention relates to the field of high-throughput sequencing, in particular to an amplicon library and a construction method thereof. Background technique [0002] The rapid acquisition of genetic information of living organisms is of great significance to the research of life sciences. The reliance of the first-generation sequencer on electrophoretic separation technology makes it difficult to further increase the speed and parallelization of analysis, and it is also difficult to reduce the cost of sequencing through miniaturization. After continuous technical development and improvement, at the beginning of the 21st century, the second-generation sequencing technology marked by Roche 454 technology, Illumina's Genome Analyzer technology and ABI's Solid technology was born. The second-generation technology has greatly reduced the cost of sequencing, and also greatly increased the speed of sequencing while maintaining high accuracy. Among...

AI Technical Summary

Problems solved by technology

When the one-step amplification library is sequenced on the machine, it can only be mixed with the amplicon library of the same variable region as one run, which not only affects the flexibility of sequencing, but also cannot guarantee the quality of sequencing

[0013] TruSeq PCR-free library construction method can directly use Illumina Miseq matching reagents for sequencing, and the biological clustering between samples is reasonable, and the repeatability of the same sample is good, but the cost of reagents is high (TruSeq DNA PCR-Free Sample Preparation Kit) , need to overcome its technical barriers

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