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High-throughput sequencing mutation detection result verifying method

A technology for detecting results and sequencing results, applied in the field of verification of high-throughput sequencing mutation detection results, which can solve problems such as false positives and missed detections

Active Publication Date: 2016-06-08
北京圣谷同创科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nevertheless, GATK software still has some missed detections and false positives in mutation search

Method used

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  • High-throughput sequencing mutation detection result verifying method
  • High-throughput sequencing mutation detection result verifying method
  • High-throughput sequencing mutation detection result verifying method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] 1. Determining the Sensitivity of High-Throughput Sequencing Mutation Detection

[0116] Design the wild-type plasmid and the mutant plasmid. The mutant plasmid contains six commonly used mutation sites. The two plasmids are mixed in proportion to form a plasmid standard product with a mutation frequency of 0.5%. The library was built and sequenced, and each sample was repeated three times, and the raw data of the sequencing were counted. The results are shown in Table 1, which shows the mutation frequency of each sample at the above mutation site.

[0117] Table 1 Standard plasmid and wild-type plasmid sequencing results

[0118]

[0119] The t test was used to analyze the difference between the frequency of each mutation site in the mutant type and the wild type, and the results showed that the p value was 1.849×10 -15 , reaching a significant difference, which proves that high-throughput sequencing can accurately measure the mutation frequency at the level of 0.5...

Embodiment 2

[0121] 1. Obtain the mutation search results of high-throughput sequencing

[0122] The cfDNA extracted from the lung cancer patient No. 1253 was subjected to IonTorrent sequencing, and VariantCallerv3.0 supporting the IonTorrent sequencing platform was used to search for mutation information. The mutations in the found EGFR gene are shown in Table 2.

[0123] Table 2 EGFR gene mutation information of cfDNA of lung cancer patient number 1253

[0124] Chrome

position

GeneSym

type

mutInfo

exon

cds_mut_syntax

aa_mut_syntax

COSM

VarFreq

Coverage

chr7

55242455

EGFR

SNPs

T>C

19

c.2225T>C

p.V742A

13183

4.06％

1108

chr7

55242470

EGFR

SNPs

T>C

19

c.2240T>C

p.L747S

26704

2.69％

930

chr7

55249054

EGFR

SNPs

C>T

20

c.2351C>T

p.S784F

13189

2.08％

240

chr7

55259515

EGFR

SNPs

T>G

21 ...

Embodiment 3

[0137] 1. Obtain the mutation search results of high-throughput sequencing

[0138] IonTorrent sequencing was carried out on the cfDNA extracted from lung cancer patients, and the mutation information was searched with VariantCallerv3.0 supporting the IonTorrent sequencing platform. ) mutations at 9 sites. VariantCallerv3.0 software reported mutation frequency as 0 for other sites.

[0139] Table 4 Mutation information found by VariantCallerv3.0

[0140]

[0141] 2. Verify the hotspot mutation

[0142] Use the following formulas a and b to verify hotspot mutations not given in the mutation search results:

[0143] Formula a: f r e q ( s n p ) = m u t _ r e a d s cov ...

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Abstract

The invention provides a verifying method of a high-throughput sequencing mutation detection result and a method for mutation searching through the high-throughput sequencing result. According to the verifying method, mathematical calculation and statistical formulas are combined, whether interested single-locus special base substitution exists in a high-throughput sequencing sample or not is determined by calculating the mutation frequency of the interested single-locus special base substitution, and whether interested continuous-locus base deletion or interested continuous-locus special base substitution exists or not is determined by calculating the variation coefficient of the interested continuous-locus base deletion or the interested continuous-locus special base substitution.

Description

technical field [0001] The invention relates to a high-throughput sequencing mutation detection result verification method. Background technique [0002] Malignant tumor is one of the major diseases threatening human health, and it is also the main cause of global morbidity and death. According to the data released by the World Health Organization, there were approximately 14 million new cancer cases and 8.2 million cancer-related deaths in 2012. According to the 2012 data released by the National Cancer Registry Center, there are about 3.5 million new cases in China every year, accounting for about one-fifth of the global incidence; the number of cancer deaths is about 2.5 million, accounting for about one-quarter of the global cancer deaths. Although my country has made great progress in tumor surgery, chemical drug treatment, radiotherapy and biotherapy, the population base makes my country the country with the highest number of cancer deaths in the world. [0003] Tumo...

Claims

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Application Information

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IPC IPC(8): G06F19/20
CPCG16B25/00
Inventor 陈威张静波
Owner 北京圣谷同创科技发展有限公司
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