Circulating tumor DNA fusion detection method based on next-generation sequencing technology

A second-generation sequencing technology and sequencing technology, which is applied in the direction of DNA preparation, recombinant DNA technology, and microbial measurement/inspection, can solve the problems of high resource requirements, low accuracy of result breakpoint coordinates, and slow detection speed, etc., to achieve increased Library conversion, good detection effect, fast running effect

Active Publication Date: 2022-02-01
BEIJING USCI MEDICAL DEVICES CO LTD
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0008] The accuracy of the breakpoint coordinates calculated by the current structural variation detection software is low. However, when performing verification experiments, it is not only necessary to know the precise breakpoint position to facilitate subsequent primer design; moreover, most of them require sequence assembly
At the same time, the current structural variation detection software still has the characteristics of slow detection speed and high resource requirements

Method used

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  • Circulating tumor DNA fusion detection method based on next-generation sequencing technology
  • Circulating tumor DNA fusion detection method based on next-generation sequencing technology
  • Circulating tumor DNA fusion detection method based on next-generation sequencing technology

Examples

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Effect test

Embodiment 1

[0083] This example provides a method for detecting fusion genes based on next-generation sequencing technology. Taking a real sample data as an example, this sample carries the classic EML4-ALK fusion, which is detected by IGV at ALK and EML4. Dot plots are as figure 1 , figure 2 shown. The specific detection method is as follows:

[0084] 1. The sample was captured and sequenced, and the sequencing strategy was PE100;

[0085] 2. Perform data preprocessing, comparison, de-duplication, and extraction of unique alignment sequences on the original off-machine data to form the final BAM file, and use Samtools to build the index of the BAM file;

[0086] 3. Extract the soft truncated parts of the sequencing reads that may carry gene fusion signals: extract the soft truncated parts of the reads whose cigar tags are "MS" or "SM" in the sequencing sequence in the BAM file according to the format rules;

[0087] 4. Split the reads into different read groups according to the coor...

Embodiment 2

[0105] This embodiment provides a method for detecting fusion genes based on next-generation sequencing technology, which is used to detect clinical peripheral blood plasma samples.

[0106] Specifically, the extraction method is as follows: 1) Pre-dissolve proteinase K in the protease dissolving buffer at a ratio of 1 mg / 550 μL to form a proteinase K solution, and add 20 μL proteinase K solution and 20 μL magnetic bead suspension to the centrifuge tube; 2 ) Transfer 300 μL of serum or plasma sample to a centrifuge tube; 3) Add 450 μL of Lysis Conjugate Solution to the centrifuge tube, vortex and mix for 30 seconds, then add 2 μL of nucleic acid sedimentation aid, and mix for 15 minutes at room temperature; 4) Transfer Put it on the magnetic stand, let it stand for 3 minutes to absorb the magnetic beads, and discard the solution; 5) Add 500 μL of the first washing solution, vortex and mix for 30 seconds; 6) Transfer to the magnetic stand, let it stand for 3 minutes to absorb th...

Embodiment 3

[0116] This embodiment provides a method for detecting fusion genes based on next-generation sequencing technology to detect mutations in low-frequency samples.

[0117] Specifically, the extraction method is as follows: 1) Pre-dissolve proteinase K in the protease dissolving buffer at a ratio of 1 mg / 550 μL to form a proteinase K solution, and add 20 μL proteinase K solution and 20 μL magnetic bead suspension to the centrifuge tube; 2 ) Transfer 300 μL of serum or plasma sample to a centrifuge tube; 3) Add 450 μL of Lysis Conjugate Solution to the centrifuge tube, vortex and mix for 30 seconds, then add 2 μL of nucleic acid sedimentation aid, and mix for 15 minutes at room temperature; 4) Transfer Put it on the magnetic stand, let it stand for 3 minutes to absorb the magnetic beads, and discard the solution; 5) Add 500 μL of the first washing solution, vortex and mix for 30 seconds; 6) Transfer to the magnetic stand, let it stand for 3 minutes to absorb the magnetic beads, and ...

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Abstract

The invention relates to the field of gene detection, in particular to a circulating tumor DNA fusion detection method based on a next-generation sequencing technology. The invention provides a gene fusion detection method based on segmented sequences, which does not involve sequence splicing in the whole calculation process, and is beneficial to saving the operation speed; and when the similarity of the sequences is compared by adopting a sequence-digital conversion method, the operation speed is higher. In addition, the invention also optimizes an extraction method and a damage repair method, which are further beneficial to the detection effect of the fusion gene.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a method for detecting fusion of circulating tumor DNA based on next-generation sequencing technology. Background technique [0002] As early as 1948, Mandel and Métais first reported the presence of free nucleotides (cfNA) in human blood. At the beginning of the report, scientists did not pay attention to the existence of cfNA. It was not until 1994 that a mutation of the RAS gene was detected in the blood of cancer patients, and people realized the importance of cfDNA. As cell-free DNA (cell-free DNA) has been detected in the blood of cancer patients with microsatellite variation, in the past ten years, researchers have studied a large number of cfNAs (DNA, mRNA, microRNAs) in the blood of cancer patients. The potential research value of cfDNA is becoming more and more significant. [0003] Compared with traditional tissue biopsy, liquid biopsy (Liquid Biopsy) has many advantage...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G16B30/10C12N15/10C12Q1/6869
CPCG16B30/10C12N15/1013C12Q1/6869
Inventor 姬晓勇汪彦荣潘晓西高司航王欢欢伍启熹王建伟
Owner BEIJING USCI MEDICAL DEVICES CO LTD
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