Validation and optimization of the Ion Torrent S5 XL sequencer and Oncomine workflow for BRCA1 and BRCA2 genetic testing

Abstract

In this study, we validated the analytical performance of BRCA1/2 sequencing using Ion Torrent's new bench-top sequencer with amplicon panel with optimized bioinformatics pipelines. Using 43 samples that were previously validated by Illumina's MiSeq platform and/or by Sanger sequencing/multiplex ligation-dependent probe amplification, we amplified the target with the Oncomine™ BRCA Research Assay and sequenced on Ion Torrent S5 XL (Thermo Fisher Scientific, Waltham, MA, USA). We compared two bioinformatics pipelines for optimal processing of S5 XL sequence data: the Torrent Suite with a plug-in Torrent Variant Caller (Thermo Fisher Scientific), and commercial NextGENe software (Softgenetics, State College, PA, USA). All expected 681 single nucleotide variants, 15 small indels, and three copy number variants were correctly called, except one common variant adjacent to a rare variant on the primer-binding site. The sensitivity, specificity, false positive rate, and accuracy for detection of single nucleotide variant and small indels of S5 XL sequencing were 99.85%, 100%, 0%, and 99.99% for the Torrent Variant Caller and 99.85%, 99.99%, 0.14%, and 99.99% for NextGENe, respectively. The reproducibility of variant calling was 100%, and the precision of variant frequency also showed good performance with coefficients of variation between 0.32 and 5.29%. We obtained highly accurate data through uniform and sufficient coverage depth over all target regions and through optimization of the bioinformatics pipeline. We confirmed that our platform is accurate and practical for diagnostic BRCA1/2 testing in a clinical laboratory.

Clinical relevance of circulating tumor DNA assessed through deep sequencing in patients with metastatic colorectal cancer

“…The clean reads were mapped to the human reference genome (hg19). Variant caller was used to filter and call the mutations in targeted regions in each gene . Cutoff level for each mutant allele frequency was defined by “variant caller” for each sample (patient).…”

“…Variant caller was used to filter and call the mutations in targeted regions in each gene. 24,25 Cutoff level for each mutant allele frequency was defined by "variant caller" for each sample (patient). The range of the limit of detection for the variants in KRAS and NRAS was from 0.05 to 0.20 in this study.…”

Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses

“…The total cost per sample when sequencing 16 samples on an Illumina MiSeq 500V2 Nano run was $76.25 and $81.07 using the Nextera XT and Kapa HyperPlus kits, respectively, compared to $129.38 and $134.20 when sequencing on a standard Illumina MiSeq 500V2 run. The cost per sample for an Ion Torrent S5 510 chip run closely matched the cost per sample of an Ion Torrent PGM 318v2 run ($124.18 and $125.04 respectively when sequencing 16 samples, Figure 6), with the S5 510 chip producing more high quality reads with a shorter run time than the PGM 318 chip (Figure 2, Table S4) (34). When comparing the Ion Torrent S5 and the Illumina MiSeq system, the difference in the cost per sample decreases with increased multiplexing.…”

The bias generated by sequencing platforms in analyzing of maternal-neonate gut microbiota profiles and diversity

“…The Ion S5-xl platform and the Hiseq 2500 platform are two commercially available sequencing platforms that target both clinical applications and laboratories [17]. There are many 16s sequencing platforms for microbiota analysis.…”