Preparation method of tumor sample sequencing reference

A reference product and sequencing technology, applied in the field of tumor sample sequencing reference product preparation, can solve the problems of high diagnostic error rate, lack of high quality, scientific and systematic comparison of results, etc., to achieve clear genetic background, good repeatability, and ensure standardization sexual effect

Active Publication Date: 2018-11-02
安徽鼎晶生物科技有限公司 +1
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Problems solved by technology

However, the clinical application of high-throughput sequencing technology is still challenging, and the high diagnostic error rate is a major problem. Continuous efforts have been made in several aspects, but the diagnostic error rate of high-throughput sequencing is still high
[0003] One of the important reasons for this phenomenon is that the quality

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  • Preparation method of tumor sample sequencing reference
  • Preparation method of tumor sample sequencing reference
  • Preparation method of tumor sample sequencing reference

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preparation example Construction

[0021] The invention provides a method for preparing a tumor sample sequencing reference product, which comprises the following steps: 1) performing monoclonal culture on tumor cell lines to obtain a monoclonal cell line; 2) extracting the whole genome of the monoclonal cell line; 3) using Perform high-throughput sequencing on the whole genome of the monoclonal cell line to determine the allele frequency to obtain the allele mutation frequency; 4) determine the dilution according to the allele mutation frequency obtained in step 3) and the target allele mutation frequency multiple; 5) after the monoclonal cell line was digested with trypsin, the cell concentration of the monoclonal cell line was obtained by counting with a flow cytometer, in terms of cell number, and the corresponding number of allele mutation frequency was 0 negative cell times Ratio diluting the monoclonal cell line to obtain mixed cells; 6) extracting the whole genome of the mixed cells in step 5), and perfo...

Embodiment 1

[0033] A new method for producing lung cancer high-throughput sequencing reference products using cell lines, comprising the following steps:

[0034] 1) Perform monoclonal culture screening of cell lines purchased from the American Type Culture Collection (ATCC), involving 8 cell lines as follows: NCI-H1650, NCI-H1975, NCI-H23, NCI-H2087, HCT-15 , LS 174T, SW1417, SW48; The monoclonal screening of cell lines adopts a dilution culture scheme, as follows: After counting cells, they are divided into 96-well plates, and the number of cells in each well is guaranteed to be 0.5, which makes single cells appear in some wells , carry out cell culture amplification on these; the final standard for determining monoclonal is to extract the genome from the cultured and expanded cells as a template, carry out high-throughput library construction, and test its allele frequency. Those with stable frequency after multiple tests are Screen qualified cell lines.

[0035] 2) The entire genome ...

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Abstract

The invention provides a preparation method of a tumor sample sequencing reference, and belongs to the technical field of high-throughput sequencing. The method comprises the following steps: performing monoclonal culture on a tumor cell line to obtain a monoclonal cell line and extracting a whole genome; determining an allele frequency; determining a dilution multiple according to an allele mutation frequency and a target allele mutation frequency; diluting the monoclonal cell line with negative cells in multiple proportion to obtain mixed cells; extracting a whole genome of the mixed cells,and determining an allele mutation frequency; and according to the comparison between the allele mutation frequency and the target allele mutation frequency of the mixed cells, refining the multiple of multiple-proportion dilution, and diluting the monoclonal cell line with negative cells to obtain the reference. According to the method disclosed by the invention, the gene background of a cell line is determined by combination of cell strain monoclone and high-throughput sequencing; and a multiple-proportion dilution method with multi-step gradual refinement is combined with a flow cytometry to accurately count cells so as to ensure high repeatability of reference preparation.

Description

technical field [0001] The invention belongs to the technical field of high-throughput sequencing, and in particular relates to a method for preparing a tumor sample sequencing reference product. Background technique [0002] High-throughput sequencing can not only clearly determine whether a disease-related gene is mutated, but also provide more quantitative information, and is increasingly accepted in clinical diagnostic applications. However, the clinical application of high-throughput sequencing technology is still challenging, and the high diagnostic error rate is a major problem. Continuous efforts have been made in several aspects, but the diagnostic error rate of high-throughput sequencing is still high. [0003] One of the important reasons for this phenomenon is that the quality of reference products used in the sequencing process is not enough to meet the requirements of clinical testing. This reason is often overlooked. Due to the lack of high-quality reference ...

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/6869
CPCC12Q1/6806C12Q1/6869C12Q2535/122
Inventor 沈伟强胡文玮孙春丽田慧焦海涛史善甫刘璐叶佳慧奚云葛海鹏陈悦科
Owner 安徽鼎晶生物科技有限公司
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