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Library construction method and application

A general-purpose sequence and amplicon library technology, applied in library construction methods and application fields, can solve the problems of primer design, complex system optimization, long manual operation time, and high requirements for library construction personnel, and achieve simple, fast and easy-to-operate library construction methods. The effect of low personnel requirements and high template capture efficiency

Active Publication Date: 2021-02-02
GENETRON HEALTH (BEIJING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are some problems in the application of the existing methods of amplification and library construction. For example, the library construction process is relatively cumbersome, requiring at least two rounds of PCR amplification and two corresponding library purifications. It takes a lot of manual operation time and requires high requirements for library construction personnel. , which is not conducive to promotion; primer design and system optimization are relatively complicated; the cost of building a library is high; the time required for the entire library building process is long and other disadvantages

Method used

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  • Library construction method and application
  • Library construction method and application
  • Library construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1. Primer design and synthesis for library construction of one-step amplicon sequencing library

[0085] 1. Primer design for one-step amplicon sequencing library construction

[0086] The present invention is used to construct the method for amplification and construction of a next-generation sequencing library, and the specific primer structures involved in the method are as follows (see figure 1 ):

[0087] Upstream outer primer F1: 5'-sequence adapter sequence 1+Barcode sequence+universal sequence-3';

[0088] Upstream inner primer F2: 5'-universal sequence+molecular tag sequence+gene upstream specific primer sequence-3';

[0089] Or upstream internal primer F2: 5'-universal sequence + gene upstream specific primer sequence-3' (molecular tags are required to detect low-frequency mutations, and molecular tags are not required to detect tissue samples);

[0090] Downstream primer R: 5'-sequence adapter sequence 2+ gene downstream specific primer sequence-3'...

Embodiment 2

[0139] Example 2. Construction and sequencing of one-step amplicon sequencing library

[0140] 1. Primer design for one-step amplicon sequencing library construction

[0141] The detection region of this experiment contains 3 amplicons (EGFR L858R, 19del and ERBB2 insertion mutations);

[0142] The test samples included 2 frozen lung cancer tissue samples (sample 1, sample 2), 4 lung cancer FFPE (formalin-fixed paraffin-embedded tissue samples) samples (sample 3, sample 4, sample 5, sample 6) and 2 White blood cell samples (sample 7, sample 8) of healthy people, the variation of the above 8 samples is known.

[0143] According to 3 amplicons (the insertion mutation of EGFR L858R, 19del and ERBB2) design the primers shown in table 3 (this embodiment adopts 8 Barcode sequences):

[0144] table 3

[0145]

[0146] Sequencing Adapters for Ion GeneStudio TM S5Plus System sequencing platform.

[0147] 2. One-step amplicon sequencing library

[0148] Nucleic acid extraction ...

Embodiment 3

[0171] Example 3. Construction and sequencing of one-step amplicon sequencing library

[0172] The samples in this experiment are plasma samples from patients with lung cancer, including plasma samples from 4 different patients and 2 healthy individuals (sample variation is known), using the kit (MagMAX TM Cell-Free DNA Isolation Kit, Applied Biosystems TM , A29319) after extracting cfDNA, library construction was performed using a primer pool with molecular tags containing insertion mutations of EGFR L858R, 19del and ERBB2.

[0173] 1. Primer design for one-step amplicon sequencing library construction

[0174] According to 3 amplicons (the insertion mutation of EGR L858R, 19del and ERBB2) design the primer shown in table 7 (upstream outer primer is identical, other is different, corresponding to 6 barcode sequences in this embodiment):

[0175] Table 7

[0176]

[0177] 2. One-step amplicon sequencing library

[0178] Nucleic acid extraction and purification kit (DNA ...

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Abstract

The invention discloses a library construction method and application. The invention provides an amplicon library construction method for detecting the variation condition of a target gene to-be-detected region of a to-be-detected sample. The method comprises the following steps: (1) designing and synthesizing an upstream outer primer F1, an upstream inner primer F2 and a downstream primer R according to a target region; and (2) performing one-step PCR amplification on a to-be-detected sample by using the upstream outer primer F1, the upstream inner primer F2 and the downstream inner primer Rto obtain an amplification product, namely an amplicon library of the target region. The one-step library construction technology can be applied to all second-generation platforms including IonTorrent, illumina and BGI / MGI, and on the basis of the library construction method, SNP, Ins / Del, CNV and methylation detection for DNA and detection products for gene fusion and expression of RNA samples are developed.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a library construction method and its application. Background technique [0002] Generally speaking, at this stage, the sequencing analysis of the target region sequence needs to be constructed first, and the way of library construction is generally nothing more than capture library construction and amplification library construction. [0003] The capture library construction is relatively speaking for the enrichment and construction of large regions of the genome, such as dozens or hundreds of gene exon regions, while the multiple amplification library construction is for specific hotspot regions or the entire exon region of individual genes. Targeted capture and sequencing analysis of exon regions. [0004] The method of amplification library construction is to design corresponding specific primers according to the target region, and then use these primers to perform...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/6858C12N15/11
CPCC40B50/06C12Q1/6858C12Q2531/113C12Q2535/122C12N15/1093C12Q1/6806C12Q1/6883C12Q2600/156C12Q2537/143C12Q2563/179C12Q2525/191C12Q1/6827C12Q1/6876C12N15/1065C12Q1/6869C12Q2600/16
Inventor 郑乔松师晓焦宇辰陈敏张凯华王思振阎海
Owner GENETRON HEALTH (BEIJING) CO LTD
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