Technical method for high-throughput construction of 16S rDNA full-length library based on Pacbio sequencing

Abstract

The invention relates to the technical field of molecular biology, and discloses a technical method for high-throughput construction of a 16S rDNA full-length library based on Pacbio sequencing, which comprises the following steps: S1, designing and synthesizing an amplification primer with a tag: the 5'end of the primer is provided with a tag sequence, the 5 'end is subjected to phosphorylation modification, the 3' end of the primer is a specific primer of an amplicon, and the specific primer is purified by HPLC (High Performance Liquid Chromatography); s2, a sample application instrument takes a chip containing 5184 micropores as a carrier, chip preparation is completed according to different flux combinations, PCR amplification is carried out, and amplification products in the chip are mixed and collected into a centrifugal tube; and S3, carrying out purification, terminal repair, A addition reaction and the like on an amplification product, and sequencing a library on a machine. A sequencing result obtained based on the method shows that the composition structure and abundance of species obtained by the method are highly consistent with those of species obtained by a conventional library building method. According to the method, the experimental operation process is greatly simplified, the method has the advantage of high throughput of gene library construction, the library construction efficiency is improved, the experimental period is shortened, and the costs of reagent consumables and the like are reduced.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a technical method for high-throughput construction of a 16S rDNA full-length library based on Pacbio sequencing. Background technique [0002] 16S rDNA is the gene encoding the small subunit of prokaryotic ribosome, with a length of about 1542bp. Its molecular size is moderate and its mutation rate is small. It is the most commonly used and useful marker in the study of bacterial taxonomy. By detecting the sequence variation and abundance of the 16S rDNA variable region, we can understand the community diversity information in environmental samples, which plays an important role in microbial taxonomic identification and microecological research. In recent years, 16S rDNA detection based on next-generation sequencing has greatly accelerated people's understanding of microbial communities. However, due to the limitation of the read length of next-generation sequencing, ...

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Problems solved by technology

[0006] Aiming at the deficiencies of the existing technology, the present invention provides a technical method for high-throughput construction of a 16S rDNA full-length library based on Pacbio sequencing, which solves the cumbersome library construction process of the conventional 16S rDNA library construction method for a large number of samples , long cycle and relatively high cost

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