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Microsatellite instability analysis method and analysis device

A microsatellite instability and instability technology, applied in the field of bioinformatics, can solve the problem of deep false positives in sequencing, and achieve the effect of high sensitivity and specificity

Pending Publication Date: 2020-12-04
QIAGEN SUZHOU TRANSLATIONAL MEDICINE CO LTD
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

Both of these methods preset the read length distribution model of microsatellite sites, which deviates from the real situation, and it is easy to cause false positives when the sequencing depth is relatively high

Method used

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  • Microsatellite instability analysis method and analysis device
  • Microsatellite instability analysis method and analysis device

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Screening of microsatellite markers

[0054] In this example, the pan-cancer NGS panel MED1CDx developed by Medtech Translational Medicine Research (Suzhou) Co., Ltd. was used to screen microsatellite markers. The MED1CDx chip was modified with gene capture probes, covering the exon regions of 601 genes. And some introns and intergenic regions, the total length is about 2.8Mb, including 23323 possible microsatellite sites with repeat sequence length ≥5 nucleotides.

[0055] Sites with single-base repeat length ≥15 nucleotides and double-base or polynucleotide repeat unit repeat times ≥8 were selected, and 138 microsatellite sites were finally obtained as MSI detection markers.

Embodiment 2

[0056] Example 2 The read length ratio distribution of different repeat lengths of microsatellite loci

[0057] (1) After the sequencing data (fastq files) of tumor samples and healthy samples are subjected to quality control filtering, they are compared to the human reference genome, and the alignment files (BAM files) of tumor samples and healthy samples are obtained respectively;

[0058] (2) Create a sequence list Lmi for each microsatellite site Mi (i=1, 2, 3, ... 138), each unit of the list is the sequence of 5 bases upstream of the microsatellite site + (microsatellite Repeat unit × n) + sequence Sn of 5 bases downstream of the microsatellite site, where n is an integer from 0 to (140÷repeat unit length);

[0059] (3) Analyze each pair of read lengths (read pair) in the BAM file of step (1), when at least one read length (read) is compared to a range of 2 kb of a microsatellite site, the read The pair is extracted, and finally the total number of reads Rmi covering the...

Embodiment 3

[0062] Example 3 Comparison of the distribution of microsatellite reads in tumor samples and healthy samples

[0063] Set the read ratio distribution of repeat sequences of different lengths at the microsatellite locus Mi in the tumor samples of the subject as Pni, and the read ratio distribution of repeat sequences of different lengths at the microsatellite locus Mi in the healthy samples as Qni, then the two Its KL divergence is:

[0064] KL(Pi||Qi)=Pni×log(Pni÷Qni)

[0065] KL(Qi||Pi)=∑Qni×log(Qni÷Pni)

[0066] The KL divergence is asymmetric, let Mi=(Pi+Qi)÷2, then the JS divergence of the two is:

[0067] JSD(Pi||Qi)=(KL(Pi||Mi)+KL(Qi||Mi))÷2

[0068] =(∑Pni×log(Pni÷(Pni+Qni))+∑Qni×log(Qni÷(Pni+Qni)))÷2+log2

[0069] The mean JSD of the subjects' 138 microsatellite loci was

[0070] AJSD=(∑JSD(Pi||Qi))÷138

[0071] Based on the accumulated data, the subject was judged to be MSI-H when AJSD ≥ 0.12.

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Abstract

The invention provides a microsatellite instability analysis method and an analysis device. The method comprises the following steps: constructing a sequence list for each screened microsatellite instability marker; obtaining a tumor sample comparison file and a health sample comparison file; respectively comparing each pair of reading lengths of the tumor sample comparison file and the health sample comparison file with a sequence in a range of 2-3kb of the microsatellite site marker to obtain a total reading number covering the microsatellite site marker; respectively comparing each pair ofreading lengths of the tumor sample comparison file and the health sample comparison file with a sequence list of the microsatellite site markers to obtain a total reading number covering the sequencelist Lmi; and calculating KL divergence and JS divergence of the tumor sample and the health sample according to the read proportion distribution. According to the method, the read length distribution between the tumor sample and the healthy sample is compared based on a robust JS divergence method, and MSI detection is accurately carried out.

Description

technical field [0001] The invention belongs to the technical field of bioinformatics, and relates to an analysis method and an analysis device for microsatellite instability. Background technique [0002] "Microsatellite" is a short tandem repeat sequence spread all over the human genome, generally with 1 to 6 bases as the repeat unit, and the number of repeats is 10 to 50 times. Compared with normal cells, due to mismatch repair (MMR) gene defects in tumor cells, the insertion or deletion of microsatellite repeat units leads to changes in the length of microsatellites, which is called microsatellite instability (MSI). MSI is closely related to the occurrence of tumors, and solid tumors with different MSI status have significantly different response rates to Keytruda and other immune checkpoint drugs. In recent years, with the approval of various immunoassay techniques, the detection of MSI / dMMR has become more and more important. [0003] Microsatellites are widely distr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G16B30/10G16B20/00
CPCG16B30/10G16B20/00
Inventor 全雪萍肖燕伟浦宇张亚飞
Owner QIAGEN SUZHOU TRANSLATIONAL MEDICINE CO LTD
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