Targeted, multiplexed PCR-based next generation sequencing (NGS) was performed on isolated DNA from each component using the Ion Ampliseq Comprehensive Cancer Panel (CCP), which targets 1,688,650 bases from 15,992 amplicons representing the complete coding sequence of 409 cancer genes (http://tools.invitrogen.com/downloads/cms_103573.csv ). Barcoded libraries were generated from 40ng of DNA per sample using the Ion AmpliSeq Comprehensive Cancer Panel (Life Technologies, Foster City, CA) and the Ion Ampliseq library kit 2.0 (Life Technologies, Foster City, CA) according to the manufacturer’s instructions with barcode incorporation. For Case 1, templates were prepared using the Ion PGM Template OT2 200 Kit (Life Technologies, Foster City, CA) on the Ion One Touch 2 according to the manufacturer’s instructions. Sequencing of multiplexed templates was performed using the Ion Torrent Personal Genome Machine (Life Technologies, Foster City, CA) on Ion 318 chips using the Ion PGM Sequencing 200 Kit v2 (200 base pair reads) according to the manufacturer’s instructions. Analysis was performed in Torrent Suite 3.6, with alignment by TMAP (version 3.6.39) using default parameters, and variant calling using the Torrent Suite Variant Caller plugin (version 3.6.63335) using default low-stringency somatic variant settings. Amplicon coverage summary files for copy number analysis were generated using the Torrent Suite Coverage Analysis plugin (v3.6.63324). Variants were annotated using Annovar [6 (link), 26 ]. For BL193A and BL193B, samples were processed, sequenced and analyzed as just described, but were assessed using a custom Ion Torrent Ampliseq panel that targets ~130 cancer related genes.
For Cases 2-4, library preparation with the CCP was performed as for Case 1. Templates were prepared using the Ion PI Template OT2 200 Kit v3 on the Ion One Touch 2 according to the manufacturer’s instructions. Sequencing of multiplexed templates was performed using the Ion Torrent Proton Sequencer using Ion Proton P1 chips using the Ion PI Sequencing 200 Kit v3 (200 base pair reads) according to the manufacturer’s instructions. Analysis was performed in Torrent Suite 4.0.2, with alignment by TMAP using default parameters, and variant calling using the Torrent Suite Variant Caller plugin (version 4.0-r76860) using default low-stringency somatic variant settings. Amplicon coverage summary files for copy number analysis were generated using the Torrent Suite Coverage Analysis plugin (v4.0-r77897).
For Cases 2-4, library preparation with the CCP was performed as for Case 1. Templates were prepared using the Ion PI Template OT2 200 Kit v3 on the Ion One Touch 2 according to the manufacturer’s instructions. Sequencing of multiplexed templates was performed using the Ion Torrent Proton Sequencer using Ion Proton P1 chips using the Ion PI Sequencing 200 Kit v3 (200 base pair reads) according to the manufacturer’s instructions. Analysis was performed in Torrent Suite 4.0.2, with alignment by TMAP using default parameters, and variant calling using the Torrent Suite Variant Caller plugin (version 4.0-r76860) using default low-stringency somatic variant settings. Amplicon coverage summary files for copy number analysis were generated using the Torrent Suite Coverage Analysis plugin (v4.0-r77897).
