Abstract 4222: Ion AmpliSeq Panel for the DNA sequencing of BRCA1 and BRCA2 genes on Ion Torrent PGM

The development of massive parallel sequencing (MPS) has revolutionized the field of genomics and created new avenues for basic research. The introduction of the benchtop sequencers enables the development of methodologies for genetic screening that are faster, more comprehensive and more cost-effective than conventional Sanger sequencing. In this study we describe a collaborative effort for the validation of a strategy for the screening of the breast cancer susceptibility genes- BRCA1 and BRCA2, making use of the most recent advances on Ion AmpliSeq™ multiplex PCR technology combined with Ion PGM™ System. Strict criteria were set to the design of the amplification primers: 1) 100% coverage of all coding exons and exon-intron boundaries; 2) amplicons covering exons must be overlapping; 3) primers must not overlap; 4) no SNPs in the last five nucleotides of primer; and 5) maximum of three non-validated SNPs per primer. Constitutional genomic DNA of 50 BRCA1 or BRCA2 mutated cases are used. The cases are divided in two groups. The first 20 cases are used to test and optimize the methodology and are selected based on the presence of mutations in or within close proximity to homopolymer regions. The additional 30 cases are used for an independent and blind evaluation and power estimation of the new methodology. All samples are studied using both “gold standard” Sanger sequencing and Ion PGM System. For data-analysis various software packages are evaluated. In the first 19 cases all SNPs and mutations, identified by Sanger sequencing were detected with our Ion AmpliSeq™ based approach. The disease-causing mutations identified included missense, nonsense and indel mutations. The number false positive results were always less than five per sample and reproducible. This work demonstrates the potential for mutational screening of BRCA1 and BRCA2 using the Ion AmpliSeq™ technology combined with the Ion Torrent PGM. Citation Format: Jose L. Costa, Arjen Mensenkamp, Alain Rico, Guoying Liu, Rosella Petraroli, Marjolijn Ligtenberg, Jose Carlos Machado. Ion AmpliSeq Panel for the DNA sequencing of BRCA1 and BRCA2 genes on Ion Torrent PGM. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4222. doi:10.1158/1538-7445.AM2013-4222