Bronchoalveolar lavage fluid sample library building method based on nanopore sequencing platform, identification method and kit

Abstract

The invention relates to a bronchoalveolar lavage fluid sample library building method based on a nanopore sequencing platform, an identification method and a kit. According to the method, based on the combination of the nanopore sequencing platform and incomplete host removal, host removal pretreatment and sample library building are carried out on metagenome samples of human bodies and animal bodies, and then used for sequencing identification of metagenomes. Compared with the traditional process, the method provided by the invention is simpler and faster, meets the reasonable detection requirements, omits the processes of library building PCR and the like in the identification process, eliminates the bias caused by PCR amplification, and can more truly and accurately identify the metagenome microorganisms.

Description

[0001] This divisional application is based on the Chinese patent application with the application number 201811528417.X, the application date is December 13, 2018, and the title of the invention is "Metagenomic sample library construction method, identification method and kit based on nanopore sequencing platform" divisional application. technical field [0002] The invention relates to the field of metagenomic identification, in particular to a method for building a library of alveolar lavage fluid samples based on a nanopore sequencing platform, an identification method and a kit. Background technique [0003] Compared with the traditional culture identification method, metagenomic sequencing identification has the advantages of short identification period and low requirements on the technical level of operators and identification personnel. Moreover, metagenomic sequencing overcomes the disadvantages of identifying many microorganisms that cannot be cultivated, and is in...

AI Technical Summary

Problems solved by technology

There are many problems in the identification of next-generation sequencing, such as: the identification of next-generation sequencing usually takes a long time from sample to result (>72h); Multi-sample combined sequencing to reduce costs; third-generation sequencing such as nanopore sequencing, with its excellent features including ultra-long sequence read length, real-time data generation, bioinformatics analysis, and compact and portable equipment, seems to be a relatively fast and convenient metagenomic research at present. method

[0007] First of all, there are some necessary purification steps in the whole wet experiment process, so the overall process is not as presented in the article and can be completed in 4 hours. According to the actual test, the whole process needs to be completed in 5.5 hours; secondly, because the host DNA step is removed as much as possible Most of the host DNA in the sample is removed, resulting in the extraction concentration of the sample is often extremely low

For example, in the process of building a sample library, due to the failure of PCR, it is impossible to continue building the library; more importantly, due to the PCR step in the library building process, the amplification bias is inevitably introduced, resulting in some bacteria with high GC content. There are serious problems in the identification of bacteria, and the proportion of bacterial species abundance is seriously unbalanced

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