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Joint element for 16S rDNA variable-zone quantitative sequencing library construction and library construction method

A technology of adapter components and sequencing libraries, applied in the field of high-throughput sequencing, can solve the problems of inability to identify bacterial strains, inability to accurately quantitatively analyze bacterial abundance, and inability to remove sequencing errors, so as to improve sequencing accuracy, avoid detection distortion, The effect of improving classification accuracy

Inactive Publication Date: 2019-05-24
武汉康测科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the problems existing in the prior art, the object of the present invention is to provide a linker element for the construction of a 16S rDNA variable region quantitative sequencing library for a high-throughput sequencing platform and a method for constructing a 16S rDNA variable region quantitative sequencing library , aims to solve a series of problems such as 16S rDNA sequencing cannot accurately quantitatively analyze bacterial abundance, cannot remove sequencing errors, and cannot identify bacterial strains

Method used

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  • Joint element for 16S rDNA variable-zone quantitative sequencing library construction and library construction method
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  • Joint element for 16S rDNA variable-zone quantitative sequencing library construction and library construction method

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Embodiment 1

[0052] The features and advantages of the present invention can be further understood from the following detailed description in conjunction with the accompanying drawings. The examples provided are merely illustrative of the methods of the present invention, and are not intended to limit the remainder of the present disclosure in any way. [Example 1] Design of adapter elements, specific targeted amplification primers, and PCR amplification primers required for the construction of 16S rDNA V4 region quantitative sequencing library

[0053] 1. One of the sequences of the linker element SEQ ID NO.1: (5'→3')GTTCAGAGTTCTACAGTCCGACGATCNNNNNNNNNNNCACTGGATACACT, wherein the italicized part is a fixed sequence FS, NNNNNNNNNN represents a variable sequence, which is composed of randomly arranged nucleotides, and N is Any base in A, T, C, G, N at different positions is the same or different base; the bold part is the hinge sequence HB; the 3' end is a protruding base T. Sequence of the...

Embodiment 2

[0070] [Example 2] 16S rDNA variable region quantitative sequencing library construction and construction process as follows figure 2 shown.

[0071] S1: Metagenomic DNA Fragmentation

[0072] 1 μg of the extracted metagenomic DNA was taken and subjected to ultrasonic fragmentation treatment (ultrasonic apparatus: Ningbo Xinzhi, JY92-IIN); 750W, 20% power, 1s ON, 1s OFF, ultrasonic for 8min.

[0073] S2: DNA end repair

[0074] Add a phosphate group at the 5' end, and add adenine A at the 3' end;

[0075] Prepare the following system:

[0076] component

Volume (μL)

End Rep mix (Vazyme, ND604-01)

7.5

Interrupted metagenomic DNA

7.4(100ng)

ddH 2 O

17.6

total

32.5

[0077] Perform the following reactions in a PCR machine

[0078] Temp

Time

20℃

30min

65℃

30min

4℃

hold

[0079] S3: Connector connection

[0080] The linker element prepared in Example 1 was ligated wit...

Embodiment 3

[0109] [Example 3] Sequencing data analysis

[0110] S1: Split the sample according to the index sequence of the sample to obtain the sequence of a single sample;

[0111] S2: Quality control of raw data to remove low-quality bases;

[0112] S3: Cluster the sequences of reads for each sample based on variable sequences (eg Figure 4 , 5 shown). Case 1: If the variable sequence VS is the same and the other partial sequences have only mismatches of less than or equal to 4 bases, then cluster all these reads into a cluster, where the number of identical reads is greater than In half of the cases, it is used as the unique sequence (unique sequence) of the cluster, and in other cases, the bases are corrected according to the base quality value to obtain the unique sequence; Case 2: If the variable sequence VS is the same and other partial sequences have more than 4 bases The mismatch of the base will be clustered into different clusters, and the sequence will be retained as the...

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Abstract

The invention discloses a joint element for 16S rDNA variable-zone quantitative sequencing library construction and a library construction method. Firstly, a double-strand DNA fragment is subjected toblunt end repair, a phosphate group is added to the 5' terminal, adenine A is added to the 3' terminal, and the joint element is connected to the processed DNA fragment through a binding reaction, wherein the joint element comprises two complementary paired DNA oligonucleotide chains, the sequence of one chain from 5' to 3' sequentially comprises a fixed sequence FS, a variable sequence VS and ahinge sequence B, the 3' terminal is a prominent basic group T, and the sequence and the other complementary sequence form a double-strand structure with one terminal provided with the single prominent basic group T. The joint element is a mixture of double-strand DNA containing variable sequences VS of different ucleotide sequences which are randomly distributed and combined. By means of the joint element, a quantitative sequencing library can be constructed from variable zones of bacterium 16S rDNA, microorganisms in samples are quantitatively detected, and identification of bacterial strains is achieved.

Description

technical field [0001] The invention belongs to the field of high-throughput sequencing, and in particular relates to a linker element for constructing a 16S rDNA variable region quantitative sequencing library for a high-throughput sequencing platform and a construction method for a 16S rDNA variable region quantitative sequencing library. Background technique [0002] The rRNA (ribosomal RNA) of prokaryotic bacteria can be divided into three types according to the sedimentation coefficient, namely 5S, 16S and 23S rRNA. 16S rRNA is a subunit of ribosomal RNA, and 16S rDNA is the gene encoding this subunit. The 16S rDNA sequence contains 10 conserved regions and 9 variable regions spaced apart from each other. The conserved regions have little difference among bacteria, while the variable regions have genus or species specificity. Using second-generation high-throughput sequencing technology to sequence the variable region can identify all bacterial species in the sample at...

Claims

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Application Information

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IPC IPC(8): C40B80/00C40B50/06G16B40/00C12Q1/6806
Inventor 吴启家王琳贾晓吴志坤
Owner 武汉康测科技有限公司
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