16S rDNA amplicon library building method for gestational diabetes mellitus intestinal flora detection

Abstract

The invention provides an amplicon library and a building method thereof, which are mainly used for detecting the distribution characteristics of intestinal flora of a pregnant woman, and performing PCR amplification on target fragments of multiple samples through intestinal excrement samples of the pregnant woman to obtain a plurality of amplification products; equivalently mixing a plurality of amplification products to obtain a mixed product; sequentially carrying out fragmentation, terminal repair and addition of 'A' to the 3' terminal on the mixed product to obtain a repair product with 'A' ; and carrying out linker connection on the repair product with the 'A' by adopting a PCR-free linker to obtain the amplicon library. By equivalently mixing of amplification products and using optimized PCR-free for linker connection, the method provided by the invention avoids preference generated by PCR and chimera in environmental samples, sequencing data of different samples is good in uniformity, subsequent clustering analysis and technical repeatability are more reasonable and accurate, the abundance distribution of intestinal flora species of pregnant women with gestational diabetes mellitus can be reflected more truly, and a rapid detection result is provided for gestational diabetes mellitus patients.

Description

technical field [0001] The invention belongs to the field of detection of intestinal flora, and in particular relates to a method for building a library of 16S rDNA amplicons for detection of intestinal flora in gestational diabetes. Background technique [0002] There are currently three main methods for library construction of the 16S V3-V4 region amplicon samples of the illumina sequencing platform: [0003] The first method is to use the earlier and extensive PCR amplification library construction method, that is, the PCR amplification step is included in the library construction process. There are two main methods for purifying and quantifying PCR amplification products carrying tag sequences by this library construction method. The first method is to use a PCR product recovery kit to purify after PCR amplification, use a spectrophotometer to quantify, and then mix equimolar amounts. This homogenization method for purification and quantification cannot remove the prim...

AI Technical Summary

Problems solved by technology

This homogenization method for purification and quantification cannot remove the primer-dimer in the PCR product, which affects the accurate quantification of the target fragment by the spectrophotometer, so that the samples cannot be truly mixed in equimolar amounts, and the uniformity of the sequencing data is poor. , the proportion of additional testing samples is high, and the project cycle cannot be guaranteed

The second method is to separate the PCR products after PCR amplification by cutting the gel to recover and quantify them with Qubit, and then mix them in equimolar amounts. This homogeneous method of purification and quantification has low gel recovery efficiency, resulting in more PCR product losses. The initial amount of database construction cannot be guaranteed to be within the normal range, which in turn affects the success rate of database construction

This method of library construction can be directly sequenced using the matching reagents of Illumina Miseq, 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), which needs to be overcome its technical limitations

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