Microbial single-cell whole genome amplification and sequencing library construction method

Abstract

The invention provides a microbial single cell whole genome amplification method which at least comprises the following steps: (1) capturing microbial single cells in a single microcavity to obtain a plurality of independent microcavities containing the microbial single cells; and carrying out microbial single-cell whole genome amplification to obtain a microbial single-cell whole genome amplification product. According to the microorganism single-cell whole genome amplification method, the volume of a reaction system is greatly reduced, the relative concentration of a substrate in the reaction system is improved, the outside and cross contamination are isolated, the microorganism single-cell whole genome amplification quality is improved, and the genome sequencing coverage rate is increased. According to the sequencing library construction method, the sequencing efficiency and the sequencing coverage rate of the genome can be greatly improved.

Description

technical field [0001] The invention relates to the field of single cell sequencing, in particular to a method for amplifying the whole genome of a single cell of microorganisms and constructing a sequencing library. Background technique [0002] For a long time, microbes remained largely a black box due to the lack of robust research methods. Since the vast majority of microorganisms cannot be pure cultured under the existing experimental conditions, the excavation and research of the microbiome mainly rely on metagenomics methods. Metagenomics takes the genomes of microbial populations in environmental samples as the research object, which essentially does not have the resolution of a single cell, and cannot obtain independent data of a single species, and the full mining of unknown microorganisms often requires the acquisition of the whole genome information of the microorganisms. Therefore, mainstream metagenomic methods are stretched in the study of rare microbial popu...

AI Technical Summary

Problems solved by technology

However, in the field of microbiological research, single-cell analysis is not popular, and the development of related methodologies is also difficult

Taking the whole genome amplification and sequencing library construction as an example, due to the small size of the microbial genome (Kbp-Mbp level), methods such as polymerase chain reaction (Polymerase chain reaction, PCR) and multiple displacement amplification (Multiple displacement amplification, MDA) were used. High-quality amplification products cannot be obtained, often accompanied by high amplification bias and loss of a large number of genomic fragments

In addition, there is still the problem of how to prepare sequencing libraries such as single-genome markers

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