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Method and test kit for determining genome instability on basis of next-generation sequencing technology

A next-generation sequencing technology and genomic technology, applied in the field of molecular detection, can solve the problems of no addition of biallelic pathogenic mutation load, low sensitivity, and poor specificity

Active Publication Date: 2020-09-18
臻和(北京)生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current existing products do not include the calculation of biallelic pathogenic mutation load, resulting in low sensitivity and poor specificity

Method used

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  • Method and test kit for determining genome instability on basis of next-generation sequencing technology
  • Method and test kit for determining genome instability on basis of next-generation sequencing technology
  • Method and test kit for determining genome instability on basis of next-generation sequencing technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1. Obtaining the Gene Sequences of the Tumor Samples and Blood Cell Samples of the Kit of the Application

[0091] 1. Fragmentation of FFPE gDNA (formalin-fixed paraffin-embedded tissue sample genomic DNA) and BC gDNA (blood sample genomic DNA)

[0092] The concentration of FFPE DNA and BC DNA was diluted to 6ng / μL. Take 55 μL for fragmentation, the fragmentation instrument is recommended to use CovarisM220 fragmentation instrument, set the equipment parameters as shown in the table below for fragmentation

[0093]

[0094] 2. Library construction

[0095] 2.1 End Repair & Add A:

[0096] 2.1.1 Configure the end repair & add A reaction system according to the table below, vortex to mix, and briefly centrifuge:

[0097]

[0098] 2.1.2 Put the configured reaction system on the PCR instrument, and perform the PCR reaction according to the table below. Note: The thermal lid temperature of the thermal cycler is set to 85°C:

[0099]

[0100] 2.2 Joint c...

Embodiment 2

[0200] Example 2. Detection of pathogenicity of homologous recombination gene in the kit of the present application

[0201] 1. Identification of homologous recombination gene SNV and indel (insert and deletion abbreviations)

[0202] Use Mutect2 software (software for identifying point mutations and insertion-deletion mutations) to analyze the results of the next-generation sequencing gene-panel comparison of tumor samples obtained in Example 1 and normal samples, and identify somatic mutations and germline mutations in tumor samples , use the annovar software (software for annotating genomic mutations) to annotate the somatic mutations and germline mutations identified by Mutect2, and those that meet the following criteria are pathogenic mutations:

[0203] 1) The number of sequences covering the mutation site is greater than 200;

[0204] 2) The mutation frequency is greater than 5%;

[0205] 3) It is recorded as a pathogenic mutation in the Clinvar database, or the mutat...

Embodiment 3

[0208] Example 3. Calculation of the mutation feature score of the kit of the present application

[0209] Somatic mutations are induced by different external or internal factors, including errors in DNA replication mechanism, induction of internal or external factors, modification of DNA modification enzymes, or failure of DNA repair enzymes. Somatic mutations caused by different factors will have different combinations of mutation types, which is the so-called mutation signature. It has been reported that Signature 3 in the mutation signature has a very strong correlation with homologous recombination pathway defects.

[0210] For the filtering results of the SNVs obtained in Example 2, the sigma software was used to calculate the score of the mutation feature related to homologous recombination.

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Abstract

The invention relates to a method and a test kit for determining genome instability on the basis of a next-generation sequencing technology, belonging to the technical field of molecular detection. Inorder to overcome the defect that genome structure variation is not considered during detection of homologous recombination defects in the prior art, the invention provides the method and the test kit for determining genome instability on the basis of the next-generation sequencing technology. The method comprises the following steps: breaking a to-be-detected gene, then adding an A connector fora corresponding PCR reaction, carrying out hybridization capture through a designed probe, then amplifying a captured DNA library, sequencing the library, and carrying out evaluating through a software so as to determine the homologous recombination defect state. Compared with the prior art, the method provided by the invention can comprehensively evaluate the HRD state, is reliable in data results and is applicable to popularization.

Description

technical field [0001] The invention relates to a method and a kit for determining genome instability based on a second-generation sequencing technology, and belongs to the technical field of molecular detection. Background technique [0002] Homologous recombination refers to the recombination between non-sister chromatids or DNA molecules containing homologous sequences on the same chromosome or within molecules. Homologous recombination allows a damaged chromosome to repair itself with the same DNA as another undamaged chromosome, which ensures the integrity of the genome. When cells have homologous recombination gene mutations, resulting in homologous recombination deficiency (homologous recombination deficiency, HRD), cells cannot repair DNA by homologous recombination. For example, the well-known breast cancer tumor-associated genes BRCA1 and BRCA2 are homologous recombination proteins. When an individual's BRCA1 and BRCA2 genes are mutated, the lifetime risk of brea...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858G16B20/50G16B40/00
CPCC12Q1/6858G16B20/50G16B40/00C12Q2531/113C12Q2535/122C12Q2549/125C12Q2537/165C12Q2521/501
Inventor 吕芳闫慧婷张亚晰郑时奇刘异倩于佳宁吕红陈维之郑杉何骥杜波
Owner 臻和(北京)生物科技有限公司
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