Low-frequency gene fusion detection method and device

A gene fusion and detection method technology, applied in biochemical cleaning devices, biochemical equipment and methods, enzymology/microbiology devices, etc., can solve problems such as difficult to obtain fusion signals, few types, and wrong distinctions

Active Publication Date: 2017-05-17
BEIJING NOVOGENE TECH CO LTD
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Problems solved by technology

[0004] The ctDNA in the blood sample is short, about 170bp, and it is difficult to obtain reliable fusion signals through double-end sequencing of both ends of the sequence, and the use of soft truncation of single-end sequences as fusion si...

Method used

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  • Low-frequency gene fusion detection method and device
  • Low-frequency gene fusion detection method and device
  • Low-frequency gene fusion detection method and device

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Embodiment approach

[0026] According to a typical implementation of the present invention, S8 includes: S81, use bwa software to compare the data after getting off the machine to the reference genome using bwa software, and use samtools software to sort and build an index; S82, compare the files Perform traversal, merge the two sequences with the same label at each site into one, and remove any inconsistent sequence compared with most other sequences in the same label; Back to the reference genome, and use samools software for sequencing and indexing. Synthesizing sequences with the same label can restore the sequence to its original condition, eliminate errors in PCR, and aligning and indexing can speed up subsequent processes.

[0027] According to a typical embodiment of the present invention, the variation detection analysis in S9 includes: S91, synthesize a long soft truncated sequence from all soft truncated soft truncated positions close to each other obtained in S83, and use the sequence ...

Embodiment 1

[0037] The samples used in this example are plasma samples from patients with non-small cell lung cancer, which are positive plasma samples for gene fusion, and the tissue RNA of the plasma samples detects EML4-ALK.E6bA20.AB374362 fusion by PCR.

[0038] Detect according to the method of the present invention below.

[0039] 1. 10ml of whole blood from patients with non-small cell lung cancer is collected and transported in BCT tubes from Streck Company. The transport temperature is room temperature and the transport time does not exceed 72 hours. Plasma was separated by two-step centrifugation, that is, centrifuged at 1600g for 10 minutes, the supernatant was taken, and then centrifuged at 16000g for 10 minutes. The supernatant was the separated plasma, which was stored at -80°C. The cfDNA in plasma was extracted using Qiagen’s circulating DNA (circulating) extraction kit, and the extracted cfDNA was stored at -20°C for later use. See Table 1 for sample names and extraction ...

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Abstract

The invention discloses a low-frequency gene fusion detection method and a low-frequency gene fusion detection device. The detection method comprises the following steps: S1, extracting cfDNA from whole blood; S2, performing tail end repairing on the cfDNA and adding A basic group at the 3' end; S3, connecting a joint containing a random label sequence; S4, designing a multiplex-PCR primer and performing target area capture; S5, performing magnetic bead purification on the PCR product in the S4, removing small fragment DNA not subjected to non-specific amplification and a primer dimer, and introducing an index sequence to obtain a ctDNA ultralow-frequency mutation library; S7, performing computer sequencing; S8, performing clustering analysis on data after library sequencing through the random label sequence; and S9, entering variation detection analysis after the quality control of the remaining data is qualified. By application of the technical scheme of the invention, the low-frequency mutation detection sensitivity is improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a detection method and device for low-frequency gene fusion. Background technique [0002] At present, the general process of next-generation sequencing to detect gene fusion of tissue samples includes: breaking genomic DNA into 300-400bp fragments, using capture probes to capture the target region, and then sequencing while synthesizing. Sequencing improves the sequencing depth under the same amount of data, and performs high sequencing depth sequencing on as much DNA as possible. When detecting fusion, the fusion signal is judged by the fact that the two sequences of double-end sequencing are not in the same region. [0003] At present, the solutions for detecting gene fusions in tissues generally use Agilent probes to capture target regions, and use illumina-Miseq or Hiseq sequencers for high-throughput sequencing. The off-machine data was compared with the reference genome usin...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12M1/34G06F19/24C12N15/10
CPCC12Q1/6806C12Q1/686C12Q1/6869G16B40/00C12Q2537/143C12Q2525/191C12Q2563/143C12Q2563/149C12Q2535/122
Inventor 朱嘉麒张振宇段楠蒋智
Owner BEIJING NOVOGENE TECH CO LTD
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