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Fast construction primer and method for water/mud sample DNA amplicon sequencing library in sewage treatment system

A sewage treatment system and sequencing library technology, applied in the field of high-throughput sequencing, can solve the problems of low quality library construction, cumbersome operation, easy pollution, etc., achieve wide application prospects, overcome low quality library construction, and easy operation

Inactive Publication Date: 2017-04-05
NANJING UNIV YIXING ENVIRONMENTAL PROTECTION RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In view of the low quality of water / mud sample library construction in the current sewage treatment system, the need to customize sequencing primers for target fragments, cumbersome operation, and high cost, the present invention designs a primer for rapid construction of an amplicon sequencing library, including adapter sequences, Index sequence, heterogeneous spacer sequence, target fragment amplification sequence, the purpose is to overcome the shortcomings of low quality, cumbersome operation, easy pollution and high cost of water / mud sample library construction in the sewage treatment system, using one-step amplification method, no need to customize The sequencing primers of the target fragments are directly sequenced using the Illumina Miseq matching kit, and the amplicon sequencing library construction method is optimized, providing a rapid construction method suitable for water / mud sample amplicon sequencing libraries in sewage treatment systems

Method used

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  • Fast construction primer and method for water/mud sample DNA amplicon sequencing library in sewage treatment system
  • Fast construction primer and method for water/mud sample DNA amplicon sequencing library in sewage treatment system
  • Fast construction primer and method for water/mud sample DNA amplicon sequencing library in sewage treatment system

Examples

Experimental program
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Effect test

Embodiment 1

[0037] (1) Extraction of DNA from water samples in sewage treatment systems

[0038] ①Collect about 2L of 500mL influent water, effluent water and three effluent samples from different treatment units of a sewage treatment plant, and filter the water samples with a 0.22um microporous membrane. The microorganisms in the water samples are enriched on the microporous membrane for use in The extraction of DNA is sequentially numbered as water sample 1, water sample 2, water sample 3, water sample 4, and water sample 5;

[0039] ②The FastDNA Soil Kit produced by MP Biomedicals in the United States was used to extract DNA from water samples, and the instruments and reagents used were prepared according to the instructions;

[0040] ③ Cut the filter membrane into pieces, put them into different sample lysis tubes, add 978 μL of sodium phosphate buffer and 122 μL of MT buffer, and crush on a biological homogenizer under the conditions of 5.5 m / s and 45 s;

[0041]④Put the broken samp...

Embodiment 2

[0074] (1) Extraction of DNA from mud samples in sewage treatment systems

[0075] Collect about 50ml of 4 mud samples from different treatment units of a sewage treatment plant, centrifuge at 10,000rpm for 10 minutes to collect about 500mg of sludge for DNA extraction; DNA extraction uses the FastDNA Soil Kit produced by MP Biomedicals in the United States, numbered as mud sample 1, Mud sample 2, mud sample 3, mud sample 4.

[0076] (2) Amplification of the target fragment

[0077] Select four primer pairs: mud sample 1 uses SEQ ID NO.4, SEQ ID NO.19; mud sample 2 uses SEQ ID NO.4, SEQ ID NO.20; mud sample 3 uses SEQ ID NO.5, SEQ ID NO .19; mud sample 4 uses SEQ ID NO.5, SEQ ID NO.20. Amplification conditions and amplification system are the same as in Example 1. Among them, in SEQ ID NO.4 and SEQ ID NO.5, bases 1-58 are linker sequences, bases 59-70 are index sequences, base 71 is a heterogeneous spacer sequence, bases 72-88 The base is the amplified sequence of the targ...

Embodiment 3

[0084] (1) Sample pretreatment:

[0085]Collect the DNA of 450 water samples and mud samples in the sewage treatment system (comprising the sample in embodiment 1, example 2), use by any one in SEQ ID NO.1~SEQ ID NO.15 and NO.16~SEQ Any 225 pairs of primers randomly combined in ID NO.30 carry out the amplification, purification, and quality identification of the target fragment (see Example 1), and each pair of primers is used twice. The size of the amplicon sequencing library of all samples In line with expectations.

[0086] (2) Mixing of amplification products:

[0087] According to the determined concentration, the samples amplified using different primers were mixed according to the same quality to obtain mixed library A and mixed library B. Each library contained 225 samples, and each library did not use repeated primer pairs.

[0088] (3) Quality identification of the mixed library:

[0089] (3a) Using Thermo Fisher's For dsDNA BR Assay Kit Reagents 2.0 fluorimet...

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Abstract

The invention discloses a fast construction primer and method for a water / mud sample DNA amplicon sequencing library in a sewage treatment system, and belongs to the field of high-throughput sequencing. The method comprises the steps of firstly, sample DNA extraction, secondly, target fragment amplification, thirdly, amplification product purification, fourthly, amplification product quality determination, fifthly, amplification product mixing, sixthly, mixed library quality determination and seventhly, mixed library preparing and sequencing. The primer comprises a connector sequence, an index sequence, a heterogeneity spacer sequence and a target fragment amplification sequence. The primer is used for further amplification, library construction can be fast completed, the sequencing cost is reduced, and a large amount of library construction time is saved. According to the fast construction primer and method, the problem about high diversity bacterial flora structure analysis in complex sewage / mud samples is solved, and the technical requirement that the fast construction primer and method are applied to water / mud sample bacterial flora deep analysis in the sewage treatment system based on the high-throughput DNA sequencing technology is met.

Description

technical field [0001] The invention belongs to the field of high-throughput sequencing, and more specifically relates to a primer and a rapid construction method for rapidly constructing a DNA amplicon sequencing library of a water / mud sample in a sewage treatment system. Background technique [0002] Currently, the mainstream next-generation sequencing platforms on the market are produced by Illumina and Life Technologies, and these sequencing platforms can generate longer read lengths. Compared with the Ion Torrent personal genome sequencer produced by Life Technologies, the Hiseq 2500, Hiseq 4000 and Miseq sequencers based on the Illumina platform can generate more data per cycle, and the advantage of the Miseq sequencer lies in the test The cycle is short, and it can quickly analyze the bacterial microbial community structure and diversity. [0003] In the bacterial genome, the rDNA gene encoding 16S rRNA has good evolutionary conservation, a length suitable for analys...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10C12Q1/68C40B50/06
CPCC12N15/11C12Q1/6806C12Q1/6869C40B50/06C12Q2531/113C12Q2535/122
Inventor 张徐祥汤俊英任洪强
Owner NANJING UNIV YIXING ENVIRONMENTAL PROTECTION RES INST
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