Microsatellite instability prediction method and application thereof

A technology of microsatellite instability and prediction method, which is applied in biochemical equipment and methods, microbial determination/inspection, genomics, etc., and can solve the problems of uneven detection standards and unsatisfactory performance.

Active Publication Date: 2020-06-19
BERRY ONCOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This puts a higher demand on the accuracy and feasibility of NGS methods, especially the performance requirements for excellent performance in panel detection are more urgent. However, most of the NGS methods reported so far are ba

Method used

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  • Microsatellite instability prediction method and application thereof
  • Microsatellite instability prediction method and application thereof
  • Microsatellite instability prediction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Collect clinical patient tissue samples, including tumor samples to be tested and their paired healthy control (normal) samples, for testing the MSI detection method of the present invention, and verify it with PCR MSI detection method, and compare with other existing technologies Known MSI detection tools for comparison. Wherein, the PCR MSI detection result is used as the gold standard to compare its consistency with the result of the MSI detection method of the present invention to evaluate the performance of the MSI detection method of the present invention. It should be noted that the MSI detection method proposed in the present invention does not require the use of control samples, which are used for PCR verification and other MSI detection software analysis in the Examples section.

[0098] As for the MSI loci used for testing, a large gene panel was used in this example for testing. The panel contained 654 genes related to tumors such as rectal cancer, gastric c...

Embodiment 2

[0101] Exon probe capture library construction sequencing was performed on the 4 gene combinations in Example 1, and a gene sequencer (NextSeq CN500) was used to perform 150bp Pair-End mode sequencing (Read1: 151, Read2: 151) according to the standard operating procedures of the instrument. ; Index1:8, Index2:8), and finally get the next-generation sequencing data in fastq format as raw data (rawdata).

[0102] The quality control software fastp was used to control the quality of the obtained next-generation sequencing data, and filter the sequencing joints, low-quality bases and sequencing error fragments, etc., and obtain high-quality data (clean data) after filtering.

[0103] Use the comparison software bwa to compare the clean data with the reference genome hg19 to obtain the corresponding specific position information on the genome of each DNA fragment; then use the gencore software to compare and de-duplicate and base-correct the data.

[0104] According to the repeat (...

Embodiment 3

[0106] According to the repeatcount result obtained in Example 2, all repeat types obtained at each site represent the allele type of the position, and for each loci, the difference between the main allele and the secondary allele abundance values ​​is calculated as the difference between the loci microsatellites Stable level prediction value, the average value of all loci in the sample (1-unstable level prediction value) is used as the microsatellite instability prediction value (MSIscore) of the sample, and the calculation formula of the non-parametric algorithm is as follows:

[0107]

[0108] Among them, X i1 Indicates the main peak, X i2 Indicates the secondary peak, i is the order of loci, and n is the number of loci.

[0109] Specifically, count the repeatcount of each detection position with a repeat length between 0-50bp, and normalize the repeatcount so that it falls within the range of 0-1. The normalization method is the reads of each repeat type at this site ...

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Abstract

The invention discloses a microsatellite instability detection and prediction method, a related site screening method, a model construction method and an application. The site screening method comprises the following steps: i) determining a microsatellite instability predicted value of a candidate microsatellite site in a sample; and/or ii) screening sites or site combinations for microsatellite instability detection by using one or more indicators including the microsatellite instability predicted value of the candidate microsatellite site; wherein the microsatellite instability predicted value is a quantified value of an abundance difference between a major allele type and a minor allele type in the candidate microsatellite site. The method provided by the invention is high in accuracy,and can accurately and stably detect the MSI state only on the basis of a single tumor sample.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and specifically relates to a method for detecting and predicting microsatellite instability, and related site screening methods, model building methods, marker combinations, kits, systems, devices, computer-readable storage media, equipment, and related methods for the quantification of microsatellite instability. Background technique [0002] Microsatellites (Microsatellites) are simple tandem repeat regions that exist in millions of loci in the human genome. Microsatellite instability (MSI) refers to the phenomenon that microsatellite sequences become longer or shorter due to DNA mismatch repair deficiency (dMMR). Such somatic mutations can cause oncogenic effects by inactivating tumor suppressor genes or disrupting other non-coding regulatory sequences. The development of MSI has become a hot spot of clinical attention, mainly due to the impact of immunotherapy on clinical practice. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858G16B20/20G16B20/30G16B40/00
CPCC12Q1/6858G16B20/20G16B20/30G16B40/00C12Q2563/159Y02A90/10
Inventor 吴书昌白健王寅
Owner BERRY ONCOLOGY CO LTD
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