Complete sequencing of the exons of BRCA1 and BRCA2 genes was performed in the Ion Torrent Personal Genome Machine (PGM) Sequencer (Thermo Fisher Scientific, Waltham, MA, USA). Sequence reads were mapped to the hg19 reference genome and used to generate BAM files; variants were predicted using the both Torrent Suit Variant Caller (TSVC, Thermo Fisher Scientific, Waltham, MA, USA) and the Genome Analysis Toolkit (GATK, Intel Corporation, Santa Clara, CA, USA).
The sequencing process started with 30 ng of DNA, which was processed according to the standard protocol Multiplex Ion AmpliSeq BRCA1 and BRCA2 Panel (Life Technologies, Carlsbad, CA, USA). The panel amplifies 167 amplicons that cover about 16.3 kb and result in coverage between 98–100% of the coding regions of both genes. The libraries were designed according to the manufacturer by the platform Ion AmpliSeq Library Preparation Protocol (Life Technologies, Carlsbad, CA, USA). Each sample was labeled individually and added to the emulsion and subsequently sequenced using a P1 chip. Each run of that protocol produced about 10 Gb of information and each sample had an average depth of 500X [27 (link)].
The sequencing process started with 30 ng of DNA, which was processed according to the standard protocol Multiplex Ion AmpliSeq BRCA1 and BRCA2 Panel (Life Technologies, Carlsbad, CA, USA). The panel amplifies 167 amplicons that cover about 16.3 kb and result in coverage between 98–100% of the coding regions of both genes. The libraries were designed according to the manufacturer by the platform Ion AmpliSeq Library Preparation Protocol (Life Technologies, Carlsbad, CA, USA). Each sample was labeled individually and added to the emulsion and subsequently sequenced using a P1 chip. Each run of that protocol produced about 10 Gb of information and each sample had an average depth of 500X [27 (link)].
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