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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 Torrent's PGM-based routine diagnostic procedure for BRCA1/2 sequencing. The procedure was first tested on a training set of 62 control samples, and then blindly validated on 77 samples in parallel with our routine technique. The training set was composed of difficult 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 specificity, an average of 1.5 confirmatory Sanger sequencings per patient was needed for complete BRCA1/2 screening. Large-scale rearrangements were identified by two distinct analyses, that is, bioinformatics and fragment analysis with electrophoresis profile 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 difficulties commonly encountered in diagnostic settings, which is why we believe our findings are of interest for the whole community, and the pipeline described can be adapted by any user of PGM for diagnostic purposes.

Introduction With 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decreased cost, capillary sequencing, which has been the norm for clinical diagnosis up until now, is becoming superseded by NGS abilities.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 shifting from Sanger sequencing platforms to higher throughput NGS platforms,,,,, already used in research. However, the performance and quality criteria expected from NGS in diagnostic and research settings are strikingly different. As an example, average coverage is usually reported in research settings, a feature irrelevant in diagnosis as the whole region of interest (ROI) must be covered, that is, 100% coverage. In other words, a diagnostic laboratory working with NGS has to provide sensitivity for its favorite genes at least equal to that of routine techniques, such as Sanger sequencing.

The Institut Curie genetic unit in Paris is actively involved in the diagnosis of breast and ovarian cancer predisposition,, with access to a high-throughput platform equipped with two Ion Torrent's Personal Genome Machine sequencers (PGM, Life Technologies, Carlsbad, CA, USA), two ABI SOLiD v4 platforms (Life Technologies) and one HiSeq (Illumina, San Diego, CA, USA). Consequently, it was tempting to test these novel technologies for diagnostic purposes in order to implement the most appropriate technology. Following pilot tests combining various enrichment procedures and sequencing platforms (see ), a PGM-based routine diagnostic procedure for BRCA1 (MIM 113705) and BRCA2 (MIM 600185) sequencing was defined and is described below. The procedure was first tested on a training set of 62 control samples, and then blindly validated on 77 samples in parallel with our routine technique.

BRCA1 and BRCA2 genes constitute a good model, as they are representative of the difficulties commonly encountered in diagnostic settings: these genes are located on autosomes, contain homopolymer regions, segmental duplication with a pseudogene located 5′ to BRCA1 and a mutational spectrum composed of all kinds of private mutations (single-nucleotide variations (SNVs), insertions/deletions (indels) and large rearrangements of one exon to the entire gene), moreover scattered throughout the coding sequence. For these reasons, we believe that our findings are of interest to the whole diagnostic community, and the diagnostic pipeline described here can be used or adapted by any user of the PGM platform for diagnostic purposes. Patients and DNA All patients attended an interview with a geneticist and a genetic counselor in a family cancer clinic at the Institut Curie, Paris, France. Matlab 2010a crack of idm download free. Genetic testing for BRCA1 and BRCA2 was proposed to women based on individual and/or family history, as previously published. Patients gave their informed consent for BRCA1/2 gene analyses. DNA was extracted using the Quickgene 610-L automated system from FujiFilm (Tokyo, Japan) according to the manufacturer's instructions and calibrated to 50 ng/μl by UV spectrophotometric assay (Nanodrop, Thermo Fischer Scientific, Waltham, MA, USA).