Amplicon next-generation sequencing based small fragment insertion and deletion detection method and device

An insertion-deletion and next-generation sequencing technology, applied in the field of biology

Active Publication Date: 2017-01-25
天津诺禾致源生物信息科技有限公司
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Problems solved by technology

However, the accuracy of the detection effect

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  • Amplicon next-generation sequencing based small fragment insertion and deletion detection method and device
  • Amplicon next-generation sequencing based small fragment insertion and deletion detection method and device
  • Amplicon next-generation sequencing based small fragment insertion and deletion detection method and device

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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 embodiment of the present invention, the method for small fragment indel detection based on amplicon next-generation sequencing is as follows: figure 1 As shown, it mainly includes two parts completed outside the small fragment indel detection program and completed within the small fragment indel 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 The background noise is obtained, and the sample data to be tested and the background n...

Embodiment 1

[0027]In this example, the sample to be tested is the NCI-H1650 cell line (purchased from the American Type Culture Collection), which is a sample of a cell line that has a 0.21% frequency of EGFR:p.E746_A750delELREA positive mutation tested by 3d-PCR.

[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]...

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Abstract

The invention discloses amplicon next-generation sequencing based small fragment insertion and deletion detection method and device. The amplicon sequencing based small fragment insertion and deletion detection method includes the steps of S1, respectively extracting DNA of a to-be-detected sample and a negative control sample, and amplifying a target region of a mutation related to the target trait by multiple amplicons; S2, carrying out the next-generation sequencing to obtain sequence of the target region; S3, comparing the sequence of the target region with a reference genome, wherein bases which are not matched with the reference genome are subjected to mispairing; S4, processing to obtain background noise of hotspot small fragment insertion and deletion related to the target trait according to the negative control sample, modeling the background noise by means of binomial distribution, distinguishing the hotspot short fragment insertion and deletion and the background noise, on each base, of the to-be-detected sample, and determining the mispairing bases as the small fragment insertion and deletion through positive detection if the proportion of the mispairing bases of the to-be-detected sample to the reference genotype is much different from that of the background noise. By the amplicon next-generation sequencing based small fragment insertion and deletion detection method and device, accuracy of small fragment insertion and deletion detection is improved.

Description

technical field [0001] The present invention relates to the field of biology, in particular to a method and device for small fragment indel detection 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...

Claims

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Application Information

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IPC IPC(8): G06F19/20
CPCG16B25/00
Inventor 朱嘉麒王棪张振宇马丽娟
Owner 天津诺禾致源生物信息科技有限公司
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