A method and kit for determining genome instability based on next-generation sequencing technology

Abstract

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. In order to overcome the lack of genome structure variation in the detection of homologous recombination defects in the prior art, the present invention provides a method and kit for determining genome instability based on next-generation sequencing technology. In this method, the gene to be tested is interrupted and A Connectors, after corresponding PCR reactions, use the designed probes for hybridization capture, amplify the captured DNA library, perform library sequencing, and then use software to evaluate the homologous recombination defect status. Compared with the existing technology, this method can comprehensively evaluate the HRD status, the data results are reliable, and it is suitable for promotion.

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...

Examples

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.

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...

Examples

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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