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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. INTRODUCTION The development of massive-parallel sequencing technology has facilitated the rapid and cost-effective generation of sequence data.

Owing to the large size of the target region of BRCA1/2 genes and no-mutation hot spots, many clinical laboratories are shifting from conventional routine techniques to high-throughput next-generation sequencing (NGS) for BRCA1/2 testing [, ]. However, diagnostic genetic testing in the clinical laboratory requires high accuracy and an acceptable turn-around time for clinical decisions. Therefore, each clinical laboratory should put in place an appropriate NGS process including the wet procedure and bioinformatics analysis that meets the quality standards of clinical genetic testing [–]. Bench-top NGS sequencers optimized for targeted sequencing have usually been evaluated for diagnostic BRCA1/2 testing [, –]. The latest Ion Torrent sequencers, models S5 and S5 XL (Thermo Fisher Scientific, Waltham, MA, USA), were released in 2015.

S5 and S5 XL require much less time for sequencing than previous Ion Torrent Personal Genome Machine (PGM) sequencer (Run time yielding 0.6–1 Gb; 2.5 hr for S5 and S5 XL vs. 4.4 hr for PGM) [, ]. Therefore, we can also expect an improvement in turn-around time required for clinical genetic testing. Several attempts have been made to compare the Illumina and Ion Torrent platforms, which adopt two different principles for library preparation and sequence generation [,, ].

The Ion Torrent platform, which uses semiconductor sequencing technology, has a reputation for higher insertion/deletion (indel) error rates associated with the homopolymer region than the Illumina platform, which sequences by synthesis technology []. However, a number of approaches including optimization of the bioinformatics pipeline, and enhancement of coverage depth and uniformity, have been attempted to overcome the shortcomings of the Ion Torrent platform [, ].

The aim of the current study was to validate and optimize the Ion Torrent S5 XL platform using the Oncomine™ BRCA Research Assay (Thermo Fisher Scientific) for routine diagnostic BRCA1/2 testing in a clinical laboratory. Furthermore, we used previously validated samples from hereditary breast and ovarian cancer syndrome (HBOC) patients for comparison of its performance with the other most commonly used platform: MiSeq sequencer with the TruSeq custom amplicon panel (both Illumina, San Diego, CA, USA) []. We also compared two variant calling methods (Torrent Variant Caller v5.2.0.34 vs NextGENe v2.4.1.2) for optimal processing of data from the Ion Torrent S5 XL.