Method for amplifying target region of nucleic acid, library building and sequencing methods and kit

A technology for target regions and nucleic acids, applied in the field of gene sequencing, can solve problems such as not reflecting the absolute abundance of species

Pending Publication Date: 2021-05-21
WUHAN BGI CLINICAL LAB CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0006] Although the current 16S amplicon library construction and sequencing technology has the advantages of high throughput, low cost, and can objectively restore the bacterial community structure and relative abundance ratio, it is the proportion of the sequence number of a certain OTU taxonomic unit to the total sequence. However, the relative abundance information cannot reflect the true absolute abundance of the species in the sample. Therefore, the results obtained by this technique say that X bacteria are more in group A than in group B. It is wrong, it should be that the abundance ratio of X bacteria in group A is higher than that in group B

Method used

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  • Method for amplifying target region of nucleic acid, library building and sequencing methods and kit
  • Method for amplifying target region of nucleic acid, library building and sequencing methods and kit
  • Method for amplifying target region of nucleic acid, library building and sequencing methods and kit

Examples

Experimental program
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Effect test

Embodiment 1

[0052] Embodiment 1 uses Tn5 transposase to carry out the method for library construction and sequencing

[0053] Embodiment 1 provides a kind of method that adopts T5 transposase to carry out library building and sequencing to the 16s DNA of microbial strain, comprises the following steps:

[0054] (1) Sample preparation: use the following four strains (subsequently referred to as A\B\C\D), extract the DNA of each strain, and then mix according to the 16S rDNA copy number of each strain, so that each microliter solution contains 50,000 copies of DNA from each strain.

[0055] Abbreviation of strain Full name of the strain GC content% copy number per μl A Rhodobacter sphaeroides 69.01 50000 B Escherichia coli 50.79 50000 C Acinetobacter baumannii 38.94 50000 D Bacillus cereus 35.58 50000

[0056] (2) Tn5 processing mark:

[0057] A. Synthesize the nucleic acid fragment containing the recognition sequence and UMI sequence (t...

Embodiment 2

[0107] Embodiment 2Tn5 reaction conditions are explored

[0108] With reference to the method of Example 1, Example 2 explored the reaction conditions of step C when step (2) Tn was used for labeling, respectively at 55 degrees Celsius, 45 degrees Celsius, 37 degrees Celsius, and 20 degrees Celsius. Down reaction 30 minutes, as shown in table 3 below:

[0109] Table 3 Amplification results and data of reaction condition exploration

[0110]

[0111]

[0112] In Table 3, the length before Tn5-UMI treatment represents: the length of the amplified fragment without using Tn5 transposase. There are certain differences in the length of Tn5-UMI before treatment obtained under different reaction conditions. The possible reasons are mainly: when using Agilent2100 for detection, the Agilent2100 instrument itself is relatively sensitive, which brings some differences in the detection results; in addition Genomic gDNA itself is a mixture of multiple bacteria, and there may also be...

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Abstract

The invention relates to the field of gene sequencing, in particular to a method for amplifying a target area of nucleic acid and library building and sequencing methods. The provided method for amplifying the target region of the nucleic acid comprises the following steps of (1) inserting a UMI sequence into the nucleic acid by using transposase so as to obtain a fusion product which contains the UMI sequence and a target region sequence; and (2) amplifying the fusion product by using a primer so as to obtain an amplification product containing the target region sequence and the UMI sequence. The invention also provides the library building method for the target region of the nucleic acid and the sequencing method. According to the method provided by the invention, the time of library building and sequencing can be shortened; and the production cost can be reduced, and the data utilization rate is effectively improved.

Description

technical field [0001] The invention relates to the field of gene sequencing, in particular to a method for amplifying a target region of nucleic acid, a method for building a library and sequencing, and a kit. Background technique [0002] Pacbio's third-generation sequencing is based on the principle of sequencing-by-synthesis, using the SMRT (single-molecule real-time fluorescent sequencing technology) chip as the carrier for the sequencing reaction. During sequencing, the genomic DNA is broken into many small fragments, and the liquid droplets are made into droplets. dispersed into different ZMW nanopores. When the polymerization reaction at the bottom of the ZMW hole occurs, the nucleotides labeled with different fluorescent lights will be retained by the polymerase in the fluorescent detection area of ​​the small hole, and the type of base composition of the template DNA can be determined according to the type of fluorescence and the duration of fluorescence. [0003]...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12N15/10C40B50/06C12Q1/6869
CPCC12Q1/6806C40B50/06C12Q1/6869C12Q2525/191
Inventor 黄标周荣芳白寅琪黄金陈智超唐冲田志坚
Owner WUHAN BGI CLINICAL LAB CO LTD
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