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Improving indel detection specificity of the Ion Torrent PGM benchtop sequencer.

Publication ,  Journal Article
Yeo, ZX; Chan, M; Yap, YS; Ang, P; Rozen, S; Lee, ASG
Published in: PLoS One
2012

The emergence of benchtop sequencers has made clinical genetic testing using next-generation sequencing more feasible. Ion Torrent's PGM™ is one such benchtop sequencer that shows clinical promise in detecting single nucleotide variations (SNVs) and microindel variations (indels). However, the large number of false positive indels caused by the high frequency of homopolymer sequencing errors has impeded PGM™'s usage for clinical genetic testing. An extensive analysis of PGM™ data from the sequencing reads of the well-characterized genome of the Escherichia coli DH10B strain and sequences of the BRCA1 and BRCA2 genes from six germline samples was done. Three commonly used variant detection tools, SAMtools, Dindel, and GATK's Unified Genotyper, all had substantial false positive rates for indels. By incorporating filters on two major measures we could dramatically improve false positive rates without sacrificing sensitivity. The two measures were: B-Allele Frequency (BAF) and VARiation of the Width of gaps and inserts (VARW) per indel position. A BAF threshold applied to indels detected by UnifiedGenotyper removed ~99% of the indel errors detected in both the DH10B and BRCA sequences. The optimum BAF threshold for BRCA sequences was determined by requiring 100% detection sensitivity and minimum false discovery rate, using variants detected from Sanger sequencing as reference. This resulted in 15 indel errors remaining, of which 7 indel errors were removed by selecting a VARW threshold of zero. VARW specific errors increased in frequency with higher read depth in the BRCA datasets, suggesting that homopolymer-associated indel errors cannot be reduced by increasing the depth of coverage. Thus, using a VARW threshold is likely to be important in reducing indel errors from data with higher coverage. In conclusion, BAF and VARW thresholds provide simple and effective filtering criteria that can improve the specificity of indel detection in PGM™ data without compromising sensitivity.

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Published In

PLoS One

DOI

EISSN

1932-6203

Publication Date

2012

Volume

7

Issue

9

Start / End Page

e45798

Location

United States

Related Subject Headings

  • Software
  • Sensitivity and Specificity
  • Polymorphism, Single Nucleotide
  • INDEL Mutation
  • Humans
  • Haploidy
  • Genome, Bacterial
  • General Science & Technology
  • Gene Frequency
  • False Positive Reactions
 

Citation

APA
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ICMJE
MLA
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Yeo, Z. X., Chan, M., Yap, Y. S., Ang, P., Rozen, S., & Lee, A. S. G. (2012). Improving indel detection specificity of the Ion Torrent PGM benchtop sequencer. PLoS One, 7(9), e45798. https://doi.org/10.1371/journal.pone.0045798
Yeo, Zhen Xuan, Maurice Chan, Yoon Sim Yap, Peter Ang, Steve Rozen, and Ann Siew Gek Lee. “Improving indel detection specificity of the Ion Torrent PGM benchtop sequencer.PLoS One 7, no. 9 (2012): e45798. https://doi.org/10.1371/journal.pone.0045798.
Yeo ZX, Chan M, Yap YS, Ang P, Rozen S, Lee ASG. Improving indel detection specificity of the Ion Torrent PGM benchtop sequencer. PLoS One. 2012;7(9):e45798.
Yeo, Zhen Xuan, et al. “Improving indel detection specificity of the Ion Torrent PGM benchtop sequencer.PLoS One, vol. 7, no. 9, 2012, p. e45798. Pubmed, doi:10.1371/journal.pone.0045798.
Yeo ZX, Chan M, Yap YS, Ang P, Rozen S, Lee ASG. Improving indel detection specificity of the Ion Torrent PGM benchtop sequencer. PLoS One. 2012;7(9):e45798.

Published In

PLoS One

DOI

EISSN

1932-6203

Publication Date

2012

Volume

7

Issue

9

Start / End Page

e45798

Location

United States

Related Subject Headings

  • Software
  • Sensitivity and Specificity
  • Polymorphism, Single Nucleotide
  • INDEL Mutation
  • Humans
  • Haploidy
  • Genome, Bacterial
  • General Science & Technology
  • Gene Frequency
  • False Positive Reactions