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High-sensitivity amplicon library building kit, library building method and application

A kit and amplicon technology, which is applied in the field of sequencing library construction, can solve the problems of labeling failure and low detection limit of library construction methods, and achieve high sensitivity

Pending Publication Date: 2021-10-01
GUANGZHOU DONGSHENG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thermofisher's amplicon library construction process also includes two rounds of PCR amplification. The first round uses specific primers with UMI for two rounds of PCR amplification to mark the template. After purification, the second step uses primers that distinguish the barcode of the sample for amplification. After purification, it is sequenced on the machine. The detection limit of this library construction method is low, and it can detect more than 0.1% of the variation, but it is easy to fail to label the less than 0.1% of the variation template.

Method used

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  • High-sensitivity amplicon library building kit, library building method and application
  • High-sensitivity amplicon library building kit, library building method and application
  • High-sensitivity amplicon library building kit, library building method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 3

[0081] Example 1 Establishment of three rounds of PCR amplification library construction method

[0082] This embodiment provides a high-sensitivity amplicon sequencing kit and usage method.

[0083] The kit includes three primer pairs: a specific primer pair for amplifying the target region; a primer pair for adding a specific molecular tag (Unique Molecular Identifier, UMI); a primer pair for adding sequencing adapters. Among them, the primers of the primer pair for adding UMI include the sequence of the amplification product of the connecting sequence, the UMI sequence and the target-specific primer pair from the 5' end to the 3' end; the primers of the primer pair for adding the sequencing adapter The sequence from the 5' end to the 3' end includes the sequence of the sequencing adapter sequence and the targeting linker sequence.

[0084] Utilize above-mentioned kit to build library, comprise the following steps (such as figure 1 shown):

[0085] (1) Obtain a DNA sampl...

Embodiment 2

[0089] Embodiment 2 Utilizes the method for building a database established in Embodiment 1 to build a database

[0090] The inventor obtained DNA samples from two individuals and performed whole exome sequencing (WES) respectively to obtain low frequency loci.

[0091] (1) The first round of PCR amplification

[0092] The variant sites shown in Table 1 were selected for targeted amplification and sequencing.

[0093] Table 1 Targeted amplification sequencing variant site information

[0094] Variation site number The chromosome where the mutation site is located start termination reference base Variant base 1 chr1 19018405 19018405 G A 2 chr1 43806166 43806166 G A 3 chr1 47746678 47746678 G C 4 chr1 65311262 65311262 G A 5 chr1 110883917 110883917 A G 6 chr1 157545384 157545384 T C

[0095] According to the mutation sites in Table 1, the target amplification region was selected and specific ...

Embodiment 3 Embodiment 2

[0160] Example 3 Sequencing and analysis of the library established in Example 2

[0161] The library constructed in Example 2 was sent to the Illumina NovaseqPE150 for sequencing and the number of 1M reads was analyzed.

[0162] Calculated by UMI2_PowerScan6 (www.genekras.com), the analysis results obtained:

[0163] As a comparison, the inventors used a two-step method to build a library (compared to Example 2, omitting the second step of using UMI tags to amplify, and the rest of the steps were the same), and using the same method to sequence the obtained library, analyze.

[0164] The library constructed in Example 2 was used for sequencing analysis, and the results are shown in Table 9.

[0165] Table 9 The sequencing analysis results of the library constructed in Example 1

[0166]

[0167] Note:

[0168] CHROM: Reference sequence name.

[0169] POS: the left-most position (1-base position) where the mutation is located, that is, the position of the first base of...

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Abstract

The invention discloses a high-sensitivity amplicon library building kit, a library building method and application, and belongs to the technical field of sequencing library building. The kit comprises: (1) at least one specific primer pair for amplifying a target area; (2) a primer pair which is used for adding a specific molecular tag UMI (Unique Molecular Identifiers); and (3) a primer pair for adding a sequencing joint. The invention further comprises the library building method by using the kit, the method comprises the following steps: carrying out a first round of PCR amplification by using the primer pair in the step (1), then carrying out a second round of PCR amplification by using the primer pair in the step (2), and finally carrying out a third round of PCR amplification by using the primer pair in the step (3), so that library building is completed, and the method can be used for mutation detection based on high-throughput sequencing. By utilizing library building, result sequencing and analysis, low-frequency mutation can be detected, the sensitivity is high, and the application is very wide.

Description

technical field [0001] The invention belongs to the technical field of sequencing library construction, and in particular relates to a high-sensitivity amplicon library construction kit and a library construction method and application. Background technique [0002] Amplicon sequencing is a highly targeted approach that allows researchers to analyze genetic variation in specific genomic regions. Ultra-deep sequencing of PCR products (amplicons) can efficiently identify and characterize variants. The method uses oligonucleotide probes to target and capture regions of interest, followed by next-generation sequencing (NGS). Amplicon sequencing offers the following advantages: a highly targeted approach allows researchers to efficiently discover, validate, and screen genetic variants; multiplex analysis of hundreds to thousands of amplicons per reaction enables high coverage Provides highly targeted multiplexing even in difficult-to-sequence regions, such as high GC regions; i...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12N15/10C40B50/06
CPCC12Q1/6806C12N15/1093C12N15/1096C40B50/06C12Q2525/191C12Q2531/113C12Q2535/122C12Q2563/179
Inventor 刘远东孟薇陈晴晴麦味子
Owner GUANGZHOU DONGSHENG BIOTECH CO LTD
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