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European Journal of Human Genetics (2014) 22, 535541& 2014 Macmillan Publishers Limited All rights reserved 1018-4813/14 http://www.nature.com/ejhg
Web End =www.nature.com/ejhg
ARTICLE
Streamlined ion torrent PGM-based diagnostics: BRCA1 and BRCA2 genes as a model
Julien Tarabeux1,2, Bruno Zeitouni3, Virginie Moncoutier1, Henrique Tenreiro1, Khadija Abidallah1, Sverine Lair3, Patricia Legoix-N4, Quentin Leroy4, Etienne Rouleau1, Lisa Golmard1,2, Emmanuel Barillot3, Marc-Henri Stern1,2, Thomas Rio-Frio4, Dominique Stoppa-Lyonnet1,2,5 and Claude Houdayer*,1,2,5
To meet challenges in terms of throughput and turnaround time, many diagnostic laboratories are shifting from Sanger sequencing to higher throughput next-generation sequencing (NGS) platforms. Bearing in mind that the performance and quality criteria expected from NGS in diagnostic or research settings are strikingly different, we have developed an Ion Torrents PGM-based routine diagnostic procedure for BRCA1/2 sequencing. The procedure was rst tested on a training setof 62 control samples, and then blindly validated on 77 samples in parallel with our routine technique. The training set was composed of difcult cases, for example, insertions and/or deletions of various sizes, large-scale rearrangements and, obviously, mutations occurring in homopolymer regions. We also compared two bioinformatic solutions in this diagnostic context, an in-house academic pipeline and the commercially available NextGene software (Softgenetics). NextGene analysis provided higher sensitivity, as four previously undetected single-nucleotide variations were found. Regarding specicity, an averageof 1.5 conrmatory Sanger sequencings per patient was needed for complete BRCA1/2 screening. Large-scale rearrangements were identied by two distinct analyses, that is, bioinformatics and fragment analysis with electrophoresis prole comparison. Turnaround time was enhanced, as a series of 30 patients were sequenced by one technician, making the results available for the clinician in 10 working days following blood sampling. BRCA1/2 genes are a good model, representative of the difculties commonly encountered in diagnostic settings, which is why we believe our ndings are of interest for the whole community, and the pipeline described can be adapted by any user of PGM for diagnostic purposes.
European Journal of Human Genetics (2014) 22, 535541; doi:http://dx.doi.org/10.1038/ejhg.2013.181
Web End =10.1038/ejhg.2013.181; published online 14 August 2013
Keywords: Ion Torrent; PGM; diagnostics; BRCA1; BRCA2
INTRODUCTIONWith progress in next-generation sequencing technologies (NGS) and a corresponding decreased cost, capillary sequencing, which has been the norm for clinical diagnosis up until now, is becoming superseded by NGS abilities. As a result, many diagnostic laboratories are...