Multiplexed sequencing library construction method

Abstract

The invention relates to the technical field of molecular biology, in particular to an ITS sequence multiplexed sequencing library construction method. The construction method includes the following steps that firstly, an amplification PCR is used, wherein a primer F1 and a primer R1 form a primer pair, and the PCR amplifies n ITS1-ITS4 sequences in a sample; secondly, a library construction PCR is used, wherein a primer F2 and a primer R2 form a primer pair, and the PCR product obtained in the first step serves as a template for PCR amplification. Quality and balance of data obtained through sequencing reaction of 40 Indexes used in the construction method are better, and sequencing results are more accurate. Marking of different library samples as many as 15*15*10 can be achieved. High-specificity amplification products can be obtained by low-complexity sample types such as animal intestinal tracts, water and yeast for making hard liquor, and high specificity is also achieved in soil, silt and other high-complexity sample types.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for constructing an ITS sequence multiple sequencing library. Background technique [0002] Fungal diversity is closely related to many fields in human life and production. Understanding and mastering the information of fungal diversity in different habitats can play an important guiding role in many aspects such as food processing, agricultural production, and medical treatment. With the development of molecular biology techniques, methods for studying fungal diversity based on ribosomal RNA (rRNA) sequencing have been continuously improved. This method extracts the microbial environmental genome (also known as metagenomics, Metagenome, that is, the sum of the genetic material of all tiny organisms in a certain habitat) in an unknown sample, amplifies the specific rRNA sequence in it by PCR, and constructs the sequencing of the specific rRNA sequence The libr...

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Problems solved by technology

For example, if the upstream PCR primers are respectively connected to 12 Indexes, and the downstream PCR primers are respectively connected to 8 Indexes, 96 library samples can be marked, that is, 20 Indexes can only be used to distinguish 96 different samples, and it is impossible to achieve Label more samples with a relatively small number of Indexes

[0006] At present, in the study of fungal diversity, ITS sequences are gradually used to replace 18S rRNA as genetic markers for the classification and identification of fungi, but the commonly used ITS primers cannot meet the needs of various sample types, such as sequencing libraries for soil samples When constructing, due to the complexity of the soil sample itself, the PCR amplification products obtained by the current conventional ITS universal primers are seriously diffused, and the amplification specificity is relatively poor

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