Point mutation detection method and device based on amplicon next-generation sequencing

A technology of next-generation sequencing and point mutation, applied in the field of biology

Active Publication Date: 2017-01-04
天津诺禾医学检验所有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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However, the accuracy of the detection effect

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  • Point mutation detection method and device based on amplicon next-generation sequencing
  • Point mutation detection method and device based on amplicon next-generation sequencing
  • Point mutation detection method and device based on amplicon next-generation sequencing

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Embodiment approach

[0022] According to a typical embodiment of the present invention, the device for obtaining mismatched bases includes: TMAP alignment software, which is used to compare the sequence of the target region with the reference genome, and form mismatches with bases that are not aligned with the reference genome. Matching; samtools software, used to establish the index of comparison files.

[0023] According to a typical implementation of the present invention, the method for detecting point mutations based on amplicon next-generation sequencing is as follows: figure 1 As shown, it mainly includes two parts completed outside the point mutation detection program and within the point mutation detection program. Among them, the former part includes sample collection, sample processing, sequencing and off-machine data; the latter includes negative control sample data processing to obtain background noise, The sample data to be tested and the background noise are detected by the two-phas...

Embodiment 1

[0027] In this example, the sample to be tested is NCI-h1975 (purchased from the American Type Culture Collection Bank), which is a sample of a cell line, and EGFR with a frequency of 0.17% was tested by 3d-pcr: c.2369C>T (T790M) positive mutation

[0028] The specific steps of this embodiment are as follows (all reagents are purchased from Thermo Fisher):

[0029] 1) Extract DNA from the sample to be tested, quantify it with a fluorometer (Qubit), concentrate or dilute with nuclease-free water to make the concentration 5ng / ul and the volume 6ul.

[0030] 2) Multiplex PCR technology was used to amplify the target region, and the PCR reaction system is shown in Table 1.

[0031] Table 1

[0032] Reagent volume High-fidelity multiplex amplification reaction mix 4μl primer mix 10μl plasma cell-free DNA 6μl

[0033] Among them, the specific sequences of the primers are shown in Table 2.

[0034] Table 2

[0035]

[0036]

[0037] The m...

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Abstract

The invention discloses a point mutation detection method and device based on amplicon next-generation sequencing. The method comprises the following steps: S1. respectively extracting DNAs of a detected sample and a negative control sample, and carrying out multiplex amplicon amplification on the target region related to target characters; S2. carrying out next-generation sequencing to obtain the sequence of the target region; S3. comparing the sequence of the target region with a reference genome, wherein unmatched bases with the reference genome form mismatches; and S4. according to the background noise of the target-character-related hotspot point mutation obtained from the negative control sample, distinguishing hotspot point mutation and background noise in the detected sample on each base by using a binomial distribution model, and if the reference genotype proportion of the mismatches bases of the detected sample is obviously different from the background noise, detecting the mismatched bases as the positive point mutation. The method and device enhance the point mutation detection accuracy based on amplicon next-generation sequencing.

Description

technical field [0001] The present invention relates to the field of biology, in particular to a method and device for detecting point mutations based on amplicon next-generation sequencing. Background technique [0002] Multiple amplicon next-generation sequencing is to customize the genomic region of interest into specific amplification primers and genomic DNA for specific amplification, enrich the DNA fragments of the target genomic region, and then use the second-generation sequencing technology for sequencing research strategy. Multiple amplicon next-generation sequencing is a hot technology in genomics research, mainly because it consumes a small amount of cost and time. At the same cost, researchers can study more samples and measure deeper depths. As a powerful and effective technology, it plays a unique role in next-generation high-throughput sequencing, and its application fields are becoming more and more extensive. [0003] Ion AmpliSeq from Thermo Fisher Scie...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12M1/00C12M1/34
CPCC12Q1/6858C12Q2537/143C12Q2535/122C12Q2537/165C12Q2545/113
Inventor 蒋智王棪朱嘉麒马丽娟师文霞
Owner 天津诺禾医学检验所有限公司
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