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.
Variant Identification Exome Sequencing Whole Exome Sequencing Torrent Variant Caller High Confident Region
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We acknowledge Prof. Massimo Gennarelli, Prof. Emilio Sacchetti, Prof. Marina Colombi and Dr. Chiara Magri for providing materials used in the study and let us include their samples data. Publication costs were covered by Grant “New Opportunities and Ways towards ERC” (NOW ERC, Project: 2014-2256) from Fondazione Cariplo and Regione Lombardia.
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Conflict of interest
The authors declare that they have no conflict of interest.
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Supplementary table 10. Detailed results on variant identification performances on the NA12878 sample, determined by comparison of WES datasets with gold standard variants provided by the GIAB consortium (XLS 11 kb)
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