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Amplicon-based semiconductor sequencing of human exomes: performance evaluation and optimization strategies.

Damiati E, Borsani G, Giacopuzzi E - Hum. Genet. (2016)

Bottom Line: Performance in variants detection was maximum at mean coverage >120×, while at 90× and 70× we measured a loss of variants of 3.2 and 4.5 %, respectively.The proposed low, medium or high-stringency filters reduced the amount of false positives by 10.2, 21.2 and 40.4 % for indels and 21.2, 41.9 and 68.2 % for SNP, respectively.False-positive variants remain an issue for the Ion Torrent technology, but our filtering strategy can be applied to reduce erroneous variants.

View Article: PubMed Central - PubMed

Affiliation: Unit of Genetics, Department of Molecular and Translational Medicine, University of Brescia, 25123, Brescia, Italy.

ABSTRACT
The Ion Proton platform allows to perform whole exome sequencing (WES) at low cost, providing rapid turnaround time and great flexibility. Products for WES on Ion Proton system include the AmpliSeq Exome kit and the recently introduced HiQ sequencing chemistry. Here, we used gold standard variants from GIAB consortium to assess the performances in variants identification, characterize the erroneous calls and develop a filtering strategy to reduce false positives. The AmpliSeq Exome kit captures a large fraction of bases (>94 %) in human CDS, ClinVar genes and ACMG genes, but with 2,041 (7 %), 449 (13 %) and 11 (19 %) genes not fully represented, respectively. Overall, 515 protein coding genes contain hard-to-sequence regions, including 90 genes from ClinVar. Performance in variants detection was maximum at mean coverage >120×, while at 90× and 70× we measured a loss of variants of 3.2 and 4.5 %, respectively. WES using HiQ chemistry showed ~71/97.5 % sensitivity, ~37/2 % FDR and ~0.66/0.98 F1 score for indels and SNPs, respectively. The proposed low, medium or high-stringency filters reduced the amount of false positives by 10.2, 21.2 and 40.4 % for indels and 21.2, 41.9 and 68.2 % for SNP, respectively. Amplicon-based WES on Ion Proton platform using HiQ chemistry emerged as a competitive approach, with improved accuracy in variants identification. False-positive variants remain an issue for the Ion Torrent technology, but our filtering strategy can be applied to reduce erroneous variants.

No MeSH data available.


Related in: MedlinePlus

Accuracy of variant identification. The performance of variant identification was assessed separately for indel (a) and SNP variants (b) by comparing the variants identified on the Ion Proton platform with the gold standard calls provided by GIAB consortium for sample NA12878. The comparison was conducted on samples sequenced using the v3 chemistry and the PI v2 chip, as well as the HiQ chemistry and the PI v3 chip. Results were also compared with those obtained using an Illumina-based exome sequencing of NA12878. The total number of variants in the GIAB and tested datasets are reported in brackets and the % of variants overlapping or unique to each compared dataset are shown in the Venn diagrams. Accuracy statistics are also reported for each comparison
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Fig2: Accuracy of variant identification. The performance of variant identification was assessed separately for indel (a) and SNP variants (b) by comparing the variants identified on the Ion Proton platform with the gold standard calls provided by GIAB consortium for sample NA12878. The comparison was conducted on samples sequenced using the v3 chemistry and the PI v2 chip, as well as the HiQ chemistry and the PI v3 chip. Results were also compared with those obtained using an Illumina-based exome sequencing of NA12878. The total number of variants in the GIAB and tested datasets are reported in brackets and the % of variants overlapping or unique to each compared dataset are shown in the Venn diagrams. Accuracy statistics are also reported for each comparison

Mentions: We estimated the accuracy of variants identification for the NA12878 reference sample by comparing the datasets of variants identified by Ion Proton platform (Table 2) with the set of gold standard variants reported by GIAB consortium. The dataset of variants from GIAB addressed by AmpliSeq Exome kit within CDS sequences was composed by 325 indels and 15,811 SNPs. The dataset produced by Ion Proton platform accounted for ~790/370 indels and ~15,250/15,800 SNPs for v3 and HiQ chemistry data, respectively. Data produced with HiQ chemistry and PI v3 chip showed a significant increase in accuracy compared to those produced using v3 chemistry and PI v2 chip. Results for SNP variants are now similar, with slightly better sensitivity, to those obtained from the Illumina dataset, while false-positive indels remain markedly higher in the HiQ dataset with and FDR value up to 47 % (Fig. 2). Detailed results on variants identification performances for each dataset are reported in Supplementary table 10.Table 2


Amplicon-based semiconductor sequencing of human exomes: performance evaluation and optimization strategies.

Damiati E, Borsani G, Giacopuzzi E - Hum. Genet. (2016)

Accuracy of variant identification. The performance of variant identification was assessed separately for indel (a) and SNP variants (b) by comparing the variants identified on the Ion Proton platform with the gold standard calls provided by GIAB consortium for sample NA12878. The comparison was conducted on samples sequenced using the v3 chemistry and the PI v2 chip, as well as the HiQ chemistry and the PI v3 chip. Results were also compared with those obtained using an Illumina-based exome sequencing of NA12878. The total number of variants in the GIAB and tested datasets are reported in brackets and the % of variants overlapping or unique to each compared dataset are shown in the Venn diagrams. Accuracy statistics are also reported for each comparison
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835520&req=5

Fig2: Accuracy of variant identification. The performance of variant identification was assessed separately for indel (a) and SNP variants (b) by comparing the variants identified on the Ion Proton platform with the gold standard calls provided by GIAB consortium for sample NA12878. The comparison was conducted on samples sequenced using the v3 chemistry and the PI v2 chip, as well as the HiQ chemistry and the PI v3 chip. Results were also compared with those obtained using an Illumina-based exome sequencing of NA12878. The total number of variants in the GIAB and tested datasets are reported in brackets and the % of variants overlapping or unique to each compared dataset are shown in the Venn diagrams. Accuracy statistics are also reported for each comparison
Mentions: We estimated the accuracy of variants identification for the NA12878 reference sample by comparing the datasets of variants identified by Ion Proton platform (Table 2) with the set of gold standard variants reported by GIAB consortium. The dataset of variants from GIAB addressed by AmpliSeq Exome kit within CDS sequences was composed by 325 indels and 15,811 SNPs. The dataset produced by Ion Proton platform accounted for ~790/370 indels and ~15,250/15,800 SNPs for v3 and HiQ chemistry data, respectively. Data produced with HiQ chemistry and PI v3 chip showed a significant increase in accuracy compared to those produced using v3 chemistry and PI v2 chip. Results for SNP variants are now similar, with slightly better sensitivity, to those obtained from the Illumina dataset, while false-positive indels remain markedly higher in the HiQ dataset with and FDR value up to 47 % (Fig. 2). Detailed results on variants identification performances for each dataset are reported in Supplementary table 10.Table 2

Bottom Line: Performance in variants detection was maximum at mean coverage >120×, while at 90× and 70× we measured a loss of variants of 3.2 and 4.5 %, respectively.The proposed low, medium or high-stringency filters reduced the amount of false positives by 10.2, 21.2 and 40.4 % for indels and 21.2, 41.9 and 68.2 % for SNP, respectively.False-positive variants remain an issue for the Ion Torrent technology, but our filtering strategy can be applied to reduce erroneous variants.

View Article: PubMed Central - PubMed

Affiliation: Unit of Genetics, Department of Molecular and Translational Medicine, University of Brescia, 25123, Brescia, Italy.

ABSTRACT
The Ion Proton platform allows to perform whole exome sequencing (WES) at low cost, providing rapid turnaround time and great flexibility. Products for WES on Ion Proton system include the AmpliSeq Exome kit and the recently introduced HiQ sequencing chemistry. Here, we used gold standard variants from GIAB consortium to assess the performances in variants identification, characterize the erroneous calls and develop a filtering strategy to reduce false positives. The AmpliSeq Exome kit captures a large fraction of bases (>94 %) in human CDS, ClinVar genes and ACMG genes, but with 2,041 (7 %), 449 (13 %) and 11 (19 %) genes not fully represented, respectively. Overall, 515 protein coding genes contain hard-to-sequence regions, including 90 genes from ClinVar. Performance in variants detection was maximum at mean coverage >120×, while at 90× and 70× we measured a loss of variants of 3.2 and 4.5 %, respectively. WES using HiQ chemistry showed ~71/97.5 % sensitivity, ~37/2 % FDR and ~0.66/0.98 F1 score for indels and SNPs, respectively. The proposed low, medium or high-stringency filters reduced the amount of false positives by 10.2, 21.2 and 40.4 % for indels and 21.2, 41.9 and 68.2 % for SNP, respectively. Amplicon-based WES on Ion Proton platform using HiQ chemistry emerged as a competitive approach, with improved accuracy in variants identification. False-positive variants remain an issue for the Ion Torrent technology, but our filtering strategy can be applied to reduce erroneous variants.

No MeSH data available.


Related in: MedlinePlus