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Tumor mutation burden standard substance, and preparation method and kit thereof

A mutation load, standard product technology, applied in biochemical equipment and methods, instruments, and microorganism determination/inspection, etc., can solve problems such as affecting TMB, and achieve accurate TMB value measurement, wide application prospects, and huge industrial application value. Effect

Active Publication Date: 2020-05-08
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, there are many problems in the detection of TMB at present, such as the calculation of TMB in the targeted genome or exome subset, whether synonymous mutations and non-synonymous mutations are included in the calculation at the same time is controversial, different sample types, processing Methods and different bioinformatics algorithms also affect the TMB value

Method used

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  • Tumor mutation burden standard substance, and preparation method and kit thereof
  • Tumor mutation burden standard substance, and preparation method and kit thereof
  • Tumor mutation burden standard substance, and preparation method and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Paired Cell Identification and Genomic DNA Extraction

[0072] The genomic DNA (genomic DNA, gDNA) of each component of the present invention is derived from a specially cultured cell line from the American type culture collection (ATCC). The corresponding list of cell lines is as follows:

[0073]

[0074] The purchased cells will be selected through monoclonal technology to select genetically stable monoclonal cell lines. In one or more embodiments of the present invention, the specific method is: when the confluence of the cells reaches 70%-90%, digest and collect the cells. To count the cells accurately, dilute the living cells to 5 / mL with medium according to the doubling dilution method, and mix the cells evenly. Take 10mL of cell suspension and distribute evenly into a 96-well plate. When cell clones are formed, monoclonal cell lines are obtained. Each cell line was identified by STR typing to confirm that the cell type was as expected, and the i...

Embodiment 2

[0091] Genomic DNA Mixing and Quality Inspection of Example 2 Paired Cell Lines

[0092] The methods of mixing and quality inspection of the test kit detection limit reference product and the repeatability reference product of the present invention are as follows:

[0093] Mix the genomic DNA of the above 6 pairs of cell lines according to different volume ratios. When mixing, the volume ratio of the genomic DNA of the tumor cell GW-PX-T (X represents any pair of the 6 pairs of cell lines) is 1% respectively ~99%, and the volume proportion of GW-PX-N genome DNA in paired leukocytes was 99%~1%, respectively. According to the analysis results of high-throughput whole exome sequencing data, each pair of cells selects a mutation site to design specific probes and primers. The sequence information is shown in the table below:

[0094]

[0095] The original mutation frequency of raw materials was confirmed by ddPCR (BIO-RAD QX200 platform). The specific ddPCR experimental proce...

Embodiment 3

[0107] Embodiment 3 reference product TMB value detection

[0108] (1) GW-P1 to GW-P6 perform high-throughput sequencing of whole exons according to the following table:

[0109]

[0110] (2) Analysis methods and parameters

[0111] Attached figure 1 The operation shown in the flow chart is as follows:

[0112] ① Data quality control: Use Fastp (https: / / github.com / OpenGene / fastp) to filter low-quality data. fastp -i read1.fastq –I read2.fastq -f 1 -F 1 -t 1 -T 1 -3 -M 25 –oread1_trimed.fastq –O read1_trimed.fastq

[0113] ② Compare the reference genome: Use BWA (http: / / bio-bwa.sourceforge.net / ) to compare the reference genome (GRCh37 / hg19)

[0114] bwa mem [-pP] [-t nThreads] [-k 32] [-w 100] [-d 100] [-r 1.5] [-A 1] [-B0] [-O 6] [-E 1] [-L 5] [-U 9] [-v 3] ref.fasta read1_trimed.fastq read1_trimed.fastq> sample1_map.sam

[0115] ③Data processing: use samtools (http: / / samtools.sourceforge.net / ) and gencore (https: / / github.com / OpenGene / gencore) to sort and remove dupli...

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Abstract

The invention provides a tumor mutation burden standard substance, and a preparation method and application thereof. The tumor mutation burden standard substance is obtained by mixing extracted genomeDNA of 6 pairs of paired cell lines according to a certain proportion. Through whole exon sequencing, a gradient reference substance is sequenced; and a tumor mutation burden value of the corresponding gradient reference substance is calculated through a specific biological information algorithm. For a detection limit reference product, a tumor cell sample is diluted by a normal cell sample in paired samples; and a droplet digital PCR method is used for verifying the dilution proportion for ensuring the mixing to conform to the expectation, so that the complexity of clinic samples and the specificity and sensitivity of an inspection and testing system are simulated to a maximum degree. The product can be used for evaluating the performance of tumor mutation burden detection products, andcan also be used for algorithm optimization or detection flow process optimization of the tumor mutation burden. The tumor mutation burden standard substance is applicable to high-flux sequencing strategies of whole exon and targeted genome or exome subsets.

Description

technical field [0001] The invention relates to the field of tumor gene mutation detection, in particular to a tumor mutation burden (Tumor Mutation Burden, TMB) standard product, a preparation method and a kit thereof. Background technique [0002] In recent years, under the vigorous promotion of national policies and the continuous replacement of technologies, molecular diagnosis, especially the field of genetic testing, has developed extremely rapidly. Genetic testing involves non-invasive prenatal testing, tumor susceptibility prediction, early diagnosis of tumors, individualized medication, and postoperative monitoring. and consumer genetic testing. Due to the huge market capacity, more and more kits for this type of genetic testing have appeared on the market in recent years. As Tumor Mutation Burden (TMB) emerges as a potential biomarker, companies in the industry are rushing to launch related tests. How to accurately measure tumor mutation load is also a major prob...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6806C12Q1/6851G16B20/50
CPCC12Q1/6806C12Q1/6851C12Q1/6886C12Q2600/156G16B20/50C12Q2531/113C12Q2563/159
Inventor 李菁华韦良慎梁达超史鹏燕林东旭
Owner 菁良科技(深圳)有限公司
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