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Homologous recombination deficiency reference material and preparation method and kit thereof

A technology of homologous recombination and reference products, applied in biochemical equipment and methods, recombinant DNA technology, DNA/RNA fragments, etc., to achieve the effect of simulating complexity and good fitting degree

Active Publication Date: 2021-06-18
菁良科技(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, how to accurately report homologous recombination repair defects has been a major problem that has plagued the related detection industry
At present, there is no suitable reference product related to homologous recombination repair on the market to help manufacturers calibrate or monitor the detection process of HRD. HRD detection urgently needs a standardized reference product to calibrate and optimize

Method used

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  • Homologous recombination deficiency reference material and preparation method and kit thereof
  • Homologous recombination deficiency reference material and preparation method and kit thereof
  • Homologous recombination deficiency reference material and preparation method and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1 Screening of cell lines, pairing cell identification and genomic DNA extraction

[0076] The genomic DNA (GDNA) of the components of the invention is derived from the cell line of American Type Culture Collection, ATCC). The corresponding list of cell lines is as follows:

[0077]

[0078] During the screening of the cell line, a very important step is to obtain a specific pair of HRD values ​​for the cell lines as a selected reference. The American Cancer Genome Spectrometer Program TCGA (The Cancer Genome Atlas) Database is only provided with sequence information of the usual cell line, does not provide HRD value information, the present invention downloads all sequence information of the TCGA database, and passes through a series of experimental operations and biology The information method is analyzed to obtain the value of HRD, so that the pair of pairing cell lines that can cover the range of 29 to 103HRD value in the TCGA database, the above-mentioned bio...

Embodiment 2

[0096] Example 2 Genomic DNA mixing and quality inspection of cell lines

[0097] The method of detection limit reference products and repetitive reference products of the present invention is as follows:

[0098] When the cell-based genomic DNA is mixed in different volume ratios, the volume ratio of tumor cell GW-PX-T (X represents 10 on the cell line) genomic DNA is 10% ~ 90%, the volume ratio of the GW-PX-N genomic DNA of the paired normal cells was 90% to 10%, respectively. According to the analysis of the high-throughput pertaining data, each pair of cells select a mutant site design specific probe and primer, using a microphone-based digital PCR (Bio-Rad QX200 platform) to the raw material original mutation frequency confirm. According to the analysis results of high-throughput pertaining data, each pair cell selection a mutant site design-specific probe and primer, genetic mutant position, primer, and probe sequence information as shown in the following table:

[0099] Gen...

Embodiment 3

[0118] Example 3 References of the reference product homologous restructuring repair defects

[0119] (1) GW-P7 ~ GW-P16 is performed in accordance with the table below:

[0120]

[0121] (2) Analytical methods and parameters

[0122] 1 Data quality control: Filter low quality data using FASTP (https: / / github.com / opengene / fastp).

[0123] 2 Comparison reference genome: use BWA (http: / / bio-bwa.sourceforge.net / ) to align the reference genome (GRCH37 / HG19)

[0124] 3 Data processing: Data uses SamTools (http: / / samtools.sourceforge.net / ) and gencore (https: / / github.com / opengene / gencore) SORT and removal DUPLICATE

[0125] 4LOH, LST and TAI for annotation and statistics scarhrd (https: / / github.com / sztup / scarhrd)

[0126] Hyrupstone deletion (LOH): 1) Lateless fragment is longer than 15MB; 2) It is shorter than the length of the entire chromosome, and one of them is counted as a LOH;

[0127] Telomeric Allelic Imalance, TAI: 1) Deleted or repeated fragment region extends to telomer;...

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Abstract

The invention discloses a homologous recombination deficiency reference material and a preparation method and kit thereof. The reference material is formed by mixing genomic DNA extracted from 10 pairs of paired cell lines according to a certain proportion, and a gradient reference product is sequenced through whole genome sequencing; a homologous recombination deficiency value of a corresponding gradient reference material is calculated through a specific biological information algorithm; while a detection limit reference product dilutes a tumor cell sample by using a normal cell sample in a paired sample, and the dilution rate is verified by using a droplet digital PCR method to guarantee that mixing meets expectation, and thus the complexity of a clinical sample and the specificity and sensitivity of an inspection and detection system are simulated to the greatest extent; and the reference material can be used for performance evaluation of a homologous recombination deficiency detection product, can also be used for algorithm optimization or detection process optimization of homologous recombination deficiency, and is suitable for sequencing strategies of high-throughput nucleotide polymorphism gene chips, targeting genomes or whole genomes.

Description

Technical field [0001] The present invention relates to the field of gene detection, and more particularly to a homologous recombination repair defect reference article and a preparation method thereof and a kit thereof. Background technique [0002] In recent years, the development of national policies has been extremely rapid, and the molecular diagnosis, especially genetic testing is extremely rapid, genetic testing involves non-invasive prenatal detection, tumor susceptibility prediction, early diagnosis of tumors, early diagnosis, individualization, postoperative monitoring As well as consumer gene detection, etc.. Due to the huge market capacity, in recent years, there have been more and more kits in such genetic assays in the market. [0003] Homologous Recombination Deficiency (HRD) has emerged as a potential biomarker, and industry companies are struggling to introduce related tests. Homologous recombination repair defects are defined as a tumor sample, and the sum of th...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6886C12Q1/686
CPCC12Q1/686C12Q1/6886C12Q2600/156C12Q2563/159
Inventor 韦良慎梁达超邱凯魏孝林刘亚胜明炳玉张秀芝李培培
Owner 菁良科技(深圳)有限公司
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