Ion ampliseq cancer hotspot panel v2

Libraries were prepared with 20 ng of genomic DNA and constructed by automated library preparation using the Ion Chef™ System and Ion Ampliseq™ Cancer Hotspot Panel v2 (Life Technologies). This panel included hotspot regions, including ~2,800 COSMIC mutations of 50 oncogenes and tumor suppressor genes with known implications in diagnosis, prognosis, and therapeutic decision-making. Regions covered and average coverage for this targeted panel are described in Table S1. DNA libraries of eight samples were combined into one library, which was then diluted to a concentration of 30 pmol/L. The diluted pooled DNA library was used for template preparation and chip loading on the Ion Chef system using Ion 520 chips, followed by sequencing on either the Ion PGM™ System or Ion S5™ Sequencer (ThermoFisher Scientific). Parameters used for assessing run quality included key signal >30, ISP loading >30%, and usable reads >30%. Parameters used for assessing sample quality included mean depth of coverage >1,000× and uniformity >90%. In some cases, samples were assessed at >500× mean coverage with adjusted cutoffs for variant reporting (VAF 15% with 500× coverage).

MDM2 amplification in baseline tumour biopsies was assessed by TaqMan quantitative PCR (Applied Biosystems), using RNAseP as the reference gene (MDM2 probe Hs01463512_cn, TaqMan Copy Number Reference Assay RNase P). Relative quantitation was performed using the ΔΔCt method and a normal healthy human donor DNA sample was used as the calibrator. Amplification was defined as having greater than five copies of MDM2 using the mean of triplicate measurements. Tumour mutation profile was assessed using the Ion AmpliSeq Cancer Hotspot Panel v2 (Life Technologies). Tumours were sequenced to a median coverage of at least × 19,000. Mutations were called using MuTect17 (link), Strelka18 (link) and SomaticIndelDetector (http://www.broadinstitute.org/cancer/cga/). Oncotator11 (link) was used to annotate mutation calls. Tumours were declared TP53 wild type if no non-synonymous mutations were called.

Target sequencing was used to compare the mutational spectrum between two metastatic liver tumours (one with SCLC and the other with adenocarcinoma) and the pre-treatment biopsy sample of the primary tumour, as described previously27 (link). Briefly, 20 ng of gDNA was used for the multiplex PCR amplification using the Ion AmpliSeq Library Kit and the Ion AmpliSeq Cancer Hotspot Panel v2 (Life Technologies) according to the manufacturer’s instructions. The Ion Xpress Barcode Adapters (Life Technologies) were ligated into the PCR products and reactions were purified with Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA). Purified libraries were pooled and sequenced on an Ion Torrent PGM device (Life Technologies) using the Ion PGM 200 Sequencing Kit v2 and the Ion 318 v2 Chip Kit. DNA sequencing data were accessed through the Torrent Suite v. 4.2 software program.

To perform mutational analysis, we extracted genomic DNA from 150,057 cells (P24) using a Qiagen kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. After the quality and quantity assessment, NGS was performed according to the instructions of Illumina (TruSeq Enrichment guide) [22 (link)]. Briefly, genomic DNA was fragmented, denatured, and hybridized. We utilized the Ion AmpliSeq™ Cancer Hotspot Panel v2 with 50 commonly oncogenes and tumor suppressor genes (Ion Torrent, Life Technologies, USA) as well as ARID1A, CCNE1, and PPP2R1B [23 (link)–27 (link)]. The captured sequences were then enriched and further amplified before being subjected to Illumina sequencing. Variant caller software was used for variant detection. Somatic nucleotide mutations were identified through the following filtering steps: (1) variant allele frequencies of > 30% and (2) sequencing coverage of > 40 reads.

PCR fragments for TP53 splice variants were amplified from cDNA of the cell lines and confirmed by sequencing as described [21] (link). The following PCR primers were used: TP53exon1_4 (forward: 5′-GGAGGAGCCGCAGTCAGAT-3′, reverse: 5′-TTCAATATCGTCCGGGGA-3′), TP53exon4_6 (forward: 5′-TCCCTTCCCAGAAAACCTACC-3′, reverse: 5′-ACCACACTATGTCGAAAAGTGTTTC-3′), TP53exon5_8 (forward: 5′-AAGCAGTCACAGCACATGAC-3′, reverse: 5-GCGGAGATTCTCTTCCTCTGT-3′), TP53exon8_10 (forward: 5′-TTTGTGCCTGTCCTGGGA-3′, reverse: 5′-TGGGCATCCTTGAGTTCC-3′) and TP53exon10_11 (forward: 5′-GAGCAGGGCTCACTCCAG-3′, reverse: 5′-TATGTCCTACTCCCCATCCTCC-3′)
Mutation screening was made using an Ion Torrent PGM sequencer with Ion 318 Chip and Life Technologies Ion AmpliSeq Cancer Hotspot Panel v2 (Life Technologies). The CHPv2.0 panel consists of 207 amplicons. The Ion AmpliSeq protocol for library preparation was used. The amplicon coordinates are documented at the vendor web site.

Target regions were amplified using the Ion AmpliSeq Cancer Hotspot Panel v2 (Life Technologies). Template DNA was prepared using the Ion PGM Hi-Q Chef Kit (Life Technologies) and sequencing was run on the Ion PGM using the Ion 314 chip. Reads were aligned to the hg19 reference and the analysis was carried out using the Ion Torrent Variant Caller Plugin and the Ion Reporter (Life Technologies).