Supplemental Methods Amaravadi Et Al

Supplemental Methods Amaravadi et al.

DNA was extracted from formalin fixed and paraffin embedded (FFPE) tissue sections of submitted polyp specimens and was sequenced by next generation sequencing (NGS)on the Ion Torrent (AmpliSeq™ Cancer Hotspot panel v.2, Life Technologies, Carlsbad, CA) and the MiSeq(Illumina TruSeq Cancer Hotspot panel, San Diego, CA) platforms.Library preparation for Ion Torrent sequencing was performed with 10 to 15 ng of input DNA and sequencingfor 50 genes performed on a 318 chip in the Ion PGM sequencer (Life Technologies, Carlsbad, CA). Library preparation for MiSeq sequencing was performed with at least 50 ng of input DNA and 47 genes were sequenced using the MiSeq reagent kit v2 (Illumina, San Diego, CA), according to the manufacturer instructions, and sequenced on the MiSeqplatform using version 2 chemistry. Ion Torrent data were analyzed with the Ion Torrent Suite v. 3.4 (Life Technologies). MiSeq sequencing data was analyzedusing a combination of commercially available bioinformatics software,and in-house bioinformatics programs and scripts (detailed in Daber et al 2013).

MiSeq sequencing

Libraries were generated for all samples using the TruSeq Cancer Amplicon Cancer Panel kit (TSCA, Illumina), covering hot-spot mutations in oncogenes and coverage across tumor suppressor genes as described (Illumina TSCA): ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, VHL. Testing was performed using between 50 and 250 ng of input DNA depending upon available amount and the degradation profile on the Genomic TapeStation (Agilent, Inc). After library preparation, samples were multiplexed and sequenced on a MiSeq (Illumina, Inc) to an average depth of coverage between 2,000-4,000x. All variants were identified using an in-house data processing bioinformatics pipeline capable of detecting single nucleotide variants, indels and gene amplification effects (12). Gene amplifications are detected informatically based on the consistency of amplicon capture with deviations from the mean only called for 4 or greatedamplicons deviating equally. These amplifications are confirmed by fluorescence in situ hybridization (FISH) studies to determine the amplification burden on a single cell basis.

All analyses were based on the human reference sequence UCSC build hg 19 (NCBI build 37.1). Single nucleotide variants with a coverage depth of <250x were excluded from the analyses. Nonsynonymous variants were reported according the Human Genome Variation Society nomenclature ( Variants found within the Exome Variant Server ( and dbSNP at a population minor allele frequency (MAF) > 0.1% were considered benign (known polymorphism) and were not reported,using an in-house analysispipeline and manual review(12).

Ion Torrent Sequencing

Libraries were generated for all samples using the Ampliseq Cancer Panel version 1 kit (Ion Torrent, Life Technologies), covering hot-spot mutations in oncogenes and coverage across tumor suppressor genes as described: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, VHL.