The Ion Torrent PGM is a semiconductor based benchtop DNA sequencer, which was launched in 2011. These studies have highlighted that the accuracy of mutation detection may need to be improved in order for NGS to become a prudent option for clinical genetic testing.
Nevertheless, several studies have compared currently available benchtop sequencers to determine their mutation detection accuracy. Compared to Sanger sequencing, benchtop NGS can detect mutations from a significantly larger number of samples in parallel, in a more cost effective manner. In recent years, the emergence of benchtop next generation sequencing (NGS) has offered a powerful alternative for mutation detection. Our findings demonstrate that although indel calling from PGM sequences may appear to be noisy at first glance, proper computational indel calling analysis is able to maximize both the sensitivity and specificity at the single base level, paving the way for the usage of this technology for future clinical genetic testing.ĭideoxynucleotide sequencing of DNA or Sanger sequencing has been the gold standard for mutation screening for over two decades. However, the variant caller of TS exhibits a lower sensitivity than GATK and SAMtools. New versions of TS, BWA and GATK have shown improvements in indel calling sensitivity and specificity over their older counterpart. In our best case scenario that involved the TMAP aligner and SAMtools, we achieved 100% sensitivity, 99.99% specificity and 29% False Discovery Rate (FDR) in indel calling from all 23 samples, which is a good performance for mutation screening using PGM.
Furthermore, we identified two variant calling measures, Quality-by-Depth (QD) and VARiation of the Width of gaps and inserts (VARW), which substantially reduced false positive indels, including non-homopolymer associated errors without compromising sensitivity. However, indel calling with the same data using the open source variant callers, GATK and SAMtools showed that false negatives could be minimised with the use of appropriate bioinformatics analysis. Our analysis revealed that false negative indels could be generated by TS under both default calling parameters and parameters adjusted for maximum sensitivity. We evaluated three major upgrades of TS by calling indels in the BRCA1 and BRCA2 genes.
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Recently, the proprietary analytical workflow for the Ion Torrent sequencer, Torrent Suite (TS), underwent a series of upgrades. Despite the PGM’s reported high accuracy in calling single nucleotide variations, it tends to generate many false positive calls in detecting insertions and deletions (indels), which may hinder its utility for clinical genetic testing. The Ion Torrent PGM is a popular benchtop sequencer that shows promise in replacing conventional Sanger sequencing as the gold standard for mutation detection.
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