High-throughput and cost-effective BRCA genetic screening is needed for application of pharmacogenetics in personalized HBOC therapy. Banerjee et al. [1] suggested that poly(ADP-ribose) polymerase (PARP) inhibitors show considerable promise for the treatment of cancers with BRCA1 or BRCA2 mutations. Secord et al. [2] demonstrated that compared to the global use of PARP inhibition, the BRCA1 and BRCA2 test for personalized PARP inhibition treatment may represent a de facto cost-reducing strategy.

In this study, results of targeted Next-generation sequencing (NGS) by Ion Torrent Personal Genome Machine (PGM, Life Technologies, Guilford, CT, USA) were compared with those of Sanger sequencing in seven HBOC patients harboring pathogenic variant or rare variant of BRCA1 and BRCA2 of uncertain clinical significance.

All enrolled subjects provided written informed consent for clinical and molecular analyses, and the study protocol was approved by the institutional review board (KC15SISE0263) of The Catholic University, Seoul, Korea. Seven HBOC patients harboring pathogenic or unclassified variants of BRCA1 and BRCA2 as confirmed by Sanger sequencing were studied. The Ion AmpliSeq BRCA1 and BRCA2 Panel (Life Technologies) consisting of 167 primer pairs in three primer pair pools was used for targeted NGS analysis. Sequencing on the Ion Torrent PGM was performed by using 500 flow runs that generated approximately 200 bp reads. Torrent Variant Caller 3.4 was applied for alignment and variant detection. The variant caller parameter setting was germline PGM high stringency (Table 1). Sequence data was visually confirmed with the Integrative Genomics Viewer (IGV) and any sequence, alignment, or variant call error artifacts were discarded. Non-synonymous variants called were evaluated by using ClinVar (http://www.ncbi.nlm.nih.gov/clinvar), the BIC database (http://research.nhgri.nih.gov/bic/), and the HGMD database (http://www.hgmd.cf.ac.uk/). Minor allele frequency (MAF) was determined from the 1000 Genomes Project database (http://www.1000genomes.org/).

Technical performance of the Ion AmpliSeq BRCA1/2 Panel showed 81% of template-positive ion sphere particle sample loading. The total read obtained was 4,541,406, with a mean read length of 137 bp. The mean sequencing depth for each region was ×494 and the average uniformity of coverage was 99%, which is percentage of target bases, covered by at least ×0.2 of the average base read depth. All variants located in all coding exons and in adjacent intronic regions of BRCA1 and BRCA2 identified by Sanger sequencing were detected by targeted NGS analysis. Direct sequencing of entire coding exons and flanking intronic sequences of BRCA1 and BRCA2 was performed on ABI 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).

Of seven patients, four carried deleterious BRCA1 or BRCA2 variants: two frameshift and two nonsense mutations. The p.Asn567Ilefs^*5 and p.Arg1203^* of BRCA1 have previously been reported in Turkish [3] and American [4] populations, respectively (Fig. 1A, B). The p.Ser1248Argfs^*10 and p.Arg2494^* of BRCA2 have been reported in German [5] and Finnish [6] populations, respectively (Fig. 1C, D). Meanwhile two BRCA1 and one BRCA2 out of nine non-synonymous variants are rare according to 1,000 Genome Project data (<1% population MAF). The p.Leu52Phe of BRCA1 and p.Val2109Ile of BRCA2 have been reported in the Koreans, and the p.Tyr856His of BRCA1 has been found in the Japanese populations.

Of various NGS platforms, the PGM generates DNA sequencing reads by detecting ions released when deoxynucleotide triphosphates are incorporated into a growing DNA strand on a semiconductor device [7]. In particular, it has been documented that indel errors occurring in homopolymer DNA regions significantly affect the specificity of indel detection owing to the nature of sequencing chemistry of PGM [8]. In this study, sequence "AGTG" at position 3972_3975 of BRCA2 was given in NGS; however, the Human Genome Variation Society notation prescribes that on the forward strand it should be "TGAG" at position 3974_3977 as given in Sanger sequencing.

While the protein-truncating variants (either frameshift, nonsense, or splice) are located generally in the coding exons or the flanking intronic sequences of BRCA genes, potentially deleterious alterations may also reside in the noncoding intronic sequences. For example, deep intronic mutation causing activation of a cryptic exon in BRCA2 has been reported in a French family with a history of breast cancer [9]. Thus, rare variants called with both NGS and Sanger sequencing should be considered for validation study, regardless of their location.

In conclusion, our study is a clear example that the quality of targeted NGS of a disease-specific subset of genes is equal to the quality of Sanger sequencing, and therefore it can be implemented reliably as a stand-alone diagnostic test demonstrated by Sikkema-Raddatz B et al. [10].