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.

Keywords: BRCA; Ion Torrent; Oncomine; S5 XL; next-generation sequencing.

Figure 1

Coverage plots from (A) 40 patients without copy number variant and (B) three representative samples showed uniform coverage across the entire region of interest. Y-axis indicates sequence read depth and x-axis indicates target

Figure 2. Copy number analysis plots from NextGENe software

Our platform successfully detected three CNVs. (A) Exon 12-14 deletion, (B) exon 4-7 deletion, and (C) exon 8-23 deletion in BRCA1.

Figure 3. A false-negative variant from a previous study [14] using the MiSeq platform and S5 XL sequencing

(A) A common single nucleotide variant (SNV) in BRCA2 (c.2971A>G, p.Asn991Asp; red solid bordered box) showed low variant frequency (12.7%) according to S5 XL sequencing owing to a rare SNV (c.3011G>A, p.Ser1044Asn; red dotted bordered box) on the primer-binding site. (B) Deep-sequencing using the other probe set revealed that the missed SNV and the adjacent rare SNV were in a cis-configuration.