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Method for analyzing tumor mutation load based on high-flux targeted sequencing

A mutation load, high-throughput technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, specific-purpose bioreactor/fermenter, etc.

Active Publication Date: 2019-10-18
GUANGZHOU BURNING ROCK DX CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, immunotherapy is not suitable for every cancer patient

Method used

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  • Method for analyzing tumor mutation load based on high-flux targeted sequencing
  • Method for analyzing tumor mutation load based on high-flux targeted sequencing
  • Method for analyzing tumor mutation load based on high-flux targeted sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1. High-throughput targeted sequencing and TMB analysis (OncoScreenPlus TMB)

[0068] 1. Pretreatment and DNA extraction of tumor tissue samples and paired control samples

[0069] In this embodiment, as the tumor tissue sample, samples with tumor cells accounting for ≥ 20% are used, and paracancerous tissue or blood leukocyte samples are selected as paired normal (control) samples. The DNA extraction process of tumor tissue samples and control samples was carried out according to the operating instructions provided with the kit (QIAamp DNA FFPE Tissue Kit, produced by QIAGEN). Subsequently, the extracted DNA was fragmented into DNA fragments with an average of about 200 bp, and used to establish a pre-library. If not used immediately for library construction, store the fragmented DNA in a -20°C freezer.

[0070] 2. Pre-library Preparation

[0071] The methodology used in the preparation of the pre-library is the classic ultrasound-interrupted double-strand l...

Embodiment 2

[0086] Example 2. TMB Detection and Evaluation of Clinical Tumor Samples

[0087] This embodiment includes 44 cases of stage IV non-small cell lung cancer samples from the top three hospitals in China (14 samples from the First Affiliated Hospital of Zhejiang University School of Medicine, and 30 samples from the Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology) and paired samples. White blood cell samples were tested for TMB. 44 samples were used to perform WES and Target capture sequencing on tumor samples using Agilent SureSelectXT HumanAll Exon V5 (detected by Mingcode (Shanghai) Biotechnology Co., Ltd.) and the method disclosed herein. Taking the gold standard WES TMB calculated by TMB as the evaluation standard, the correlation between TMB (OncoScreenPlus TMB) calculated by the disclosed method and WES TMB = 0.841, R2 = 0.707, such as figure 2 shown. The TMB cut-off value reported in the literature is 7.4 [1] , 9.65...

Embodiment 3I

[0090] Example 3.In silico TMB detection and evaluation

[0091]This example adopts in silico methodology to evaluate the consistency between OncoScreenPlus TMB and WES TMB. Specifically, the data MAF files of TCGA MC3 samples, including 32 cancers, were downloaded from the database website of the National Institutes of Health (NIH) (https: / / gdc.cancer.gov / about-data / publications / mc3-2017) The mutation data of 8291 cases of samples are shown in Table 3. Refer to the FoCR (Friends of Cancer Research) WES TMB standard to calculate the WES TMB: (1) FILTER! ="PASS" mutations are defined as false positives (variant artifacts), remove samples containing more than 50% of false positive mutations; (2) remove false positive mutations in samples; (3) remove mutation sequences (variantcount)<3 Mutation; (4) remove mutations with allele frequency (allele frequency) <5%; (5) remove mutations with total depth (total depth) <25; (6) select mutations in coding regions (about 32.3Mb) in CCDS...

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Abstract

The invention relates to a method and system for analyzing a tumor mutation load based on high-flux targeted sequencing data. The method comprises the following steps that through high-flux targeted sequencing, sequences of selected exons and upstream and downstream areas of target genes in a tumor sample and a pairing contrast sample are subjected to sequencing; sequencing data obtained from thetumor sample and the pairing contrast sample is compared with a human reference genome sequence, and the number of mutated somatic cells in the sequences of the selected exons and the upstream and downstream areas of the target genes in the tumor sample is calculated; the mutation numbers of the somatic cells in the selected exons and the upstream and downstream areas of the target genes are addedto obtain a total mutation number, the lengths of the selected exons and the upstream and downstream areas of the target genes are added to obtain a total area length (Mb), and TMB is calculated through the following formula: TMB=the total mutation number / the total area length.

Description

technical field [0001] The disclosure belongs to the field of gene detection in bioinformatics technology, and in particular relates to a method and system for analyzing tumor mutation load by using high-throughput targeted sequencing data. [0002] technical background [0003] Chemotherapy and radiotherapy, targeted therapy, and immunotherapy are listed as three revolutions in cancer treatment. Among them, immunotherapy has attracted widespread attention from scientists, doctors and patients due to its advantages of low toxicity, strengthening the immune system without damaging it, and reducing the recurrence rate of cancer. In recent years, immunotherapy has shown remarkable clinical efficacy in the treatment of melanoma, non-small cell lung cancer, head and neck cancer, urothelial cancer, and other tumors with mismatch repair deficiency. Immunological drugs, such as O drug (Opdivo) and K drug (Keytruda) recently approved for marketing in China, as well as domestically pr...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12M1/00C12M1/34
CPCC12Q1/6858C12Q2535/122C12Q2537/165
Inventor 张周张之宏侯婷李冰揣少坤汉雨生
Owner GUANGZHOU BURNING ROCK DX CO LTD
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