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Method for constructing amplicon library

A technology of amplicon library and construction method, which is applied in the direction of chemical library, biochemical equipment and method, microorganism measurement/inspection, etc. It can solve the problems of many non-specific bands and low qualification rate, and achieve non-specific bands Less, improve the pass rate, reduce the effect of volatility

Active Publication Date: 2015-04-29
BEIJING NOVOGENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using the existing amplification technology to amplify the above-mentioned amplicons, the qualified rate is low and there are many non-specific bands

Method used

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  • Method for constructing amplicon library
  • Method for constructing amplicon library
  • Method for constructing amplicon library

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: A kind of method of 16SV4 amplicon library construction, concrete steps are as follows:

[0041] (1) Collect environmental microbial samples and extract genomic DNA from the samples. For example, using the Soil Genomic DNA Extraction Kit (DP336, Tiangen) to extract microbial DNA from soil samples.

[0042] (2) Use agarose gel electrophoresis to detect the integrity, concentration and contamination of genomic DNA. If there is a large amount of RNA in the sample, it needs to be digested with RNase. If there are many impurities in the sample (such as humic acid, organic solvent, etc.), XP magnetic beads can be used to purify the sample, and the sample can be accurately quantified in combination with a spectrophotometer or Qubit .

[0043] (3) According to the DNA concentration of the sample, the sample is diluted to reach an appropriate concentration.

[0044] (4) Amplify the hypervariable region V4 of 16S rDNA (use primers SEQ ID NO: 1: 16SV4-F-GTGCCAGCMG...

Embodiment 2

[0052] Embodiment 2: A method for constructing an ITS1 amplicon library, the main experimental steps are as follows:

[0053] (1) Collect environmental microbial samples and extract genomic DNA from the samples. For example, using the Soil Genomic DNA Extraction Kit (DP336, Tiangen) to extract microbial DNA from soil samples.

[0054] (2) Use agarose gel electrophoresis to detect the integrity, concentration and contamination of genomic DNA. If there is a large amount of RNA in the sample, it needs to be digested with RNase; if there are many impurities in the sample (such as humic acid, organic solvent, etc.), the sample can be purified using XP magnetic beads; at the same time, the sample can be accurately quantified by combining a spectrophotometer or Qubit .

[0055] (3) According to the DNA concentration of the sample, the sample is diluted to reach an appropriate concentration.

[0056] (4) Amplify ITS1 (use primers SEQ ID NO: 3: ITS1-F-GGAAGTAAAAGTCGTAACAAGG; SEQ ID ...

Embodiment 3

[0064] Embodiment 3: A method applied to the construction of ITS2 amplicon library, the main experimental steps are as follows:

[0065](1) Collect environmental microbial samples and extract genomic DNA from the samples. For example, using the Soil Genomic DNA Extraction Kit (DP336, Tiangen) to extract microbial DNA from soil samples.

[0066] (2) Use agarose gel electrophoresis to detect the integrity, concentration and contamination of genomic DNA. If there is a large amount of RNA in the sample, it needs to be digested with RNase; if there are many impurities in the sample (such as humic acid, organic solvent, etc.), the sample can be purified with XP magnetic beads. At the same time, it can be combined with a spectrophotometer or Qubit to accurately quantify the sample.

[0067] (3) According to the DNA concentration of the sample, the sample is diluted to reach an appropriate concentration.

[0068] (4) For ITS2 (use primers SEQ ID NO:5: ITS2-F-GCATCGCATAAGAACGCAGC; S...

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Abstract

The invention discloses a method for constructing an amplicon library. The method comprises the following steps: S1, amplifying a destination fragment to obtain enriched destination fragments; S2, performing fragmented library construction on the enriched destination fragments to obtain the amplicon library, wherein the step S1 comprises the following sub-steps: S11, quantifying an amplification template of the destination fragment; S12, fixing annealing temperature of an amplification primer of the destination fragment; S13, amplifying the destination fragment at the annealing temperature by utilizing the amplification primer to obtain the enriched destination fragments. According to the constructing method, the template amount in an amplification system is accurately quantified for nucleic acid samples from different environments, so that the amplification efficiency of destination fragments of different samples are enabled to be the same and the fluctuation property of sequencing data is reduced; the annealing temperature of the used primer is optimized, so that the proportion of non-specific amplicons in the amplicon library is reduced and the qualification rate of the amplicon library is increased.

Description

technical field [0001] The present invention relates to the field of high-throughput sequencing, in particular to a method for constructing an amplicon library. Background technique [0002] The rapid acquisition of genetic information of living organisms is of great significance to the research of life sciences. The reliance of the first-generation sequencer on electrophoretic separation technology makes it difficult to further increase the speed and parallelization of analysis, and it is also difficult to reduce the cost of sequencing through miniaturization. After continuous technical development and improvement, at the beginning of the 21st century, the second-generation sequencing technology marked by Roche 454 technology, Illumina's Genome Analyzer technology and ABI's Solid technology was born. The second-generation technology has greatly reduced the cost of sequencing, and also greatly increased the speed of sequencing while maintaining high accuracy. Among them, I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/68
Inventor 王大伟刘运超蒋智李明渊朱海浩胡政
Owner BEIJING NOVOGENE TECH CO LTD
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