Surecall software

Raw fastq files were processed with the Agilent SureCall Software (version 3.5.1.46). A median sequencing depth of 158-fold, 83-fold, 74-fold, and 47-fold was achieved in germline, FFPE, cfDNA, and CTC samples, respectively. Personal alterations were excluded when detected in the individual whole blood sample. Remaining alterations were sieved based on their predicted deleterious effect annotated in the COSMIC database [51 (link), 52 ] and the Cancer Genome Interpreter [53 (link), 54 ]. Further analysis was performed as previously described [50 (link)]. The detailed procedure of variant calling and data analysis is described in the Supplementary information and depicted in Supplementary Fig. 2. Sequencing data will be available from the corresponding author upon reasonable request.

Targeted sequencing was performed using the HaloPlex Target Enrichment System for Illumina sequencing (Agilent Technologies, Santa Clara, CA, USA) for a custom-designed set of 812 cancer-related genes. Data were analysed by SureCall software (version 2.0.7.0, Agilent Technologies) and Integrative Genomics Viewer (IGV) (version 2.3.25, Broad Institute). Targeted highly multiplexed PCR with semiconductor-based sequencing using the Ion AmpliSeq assay was performed as described previously15 (link), analysing the MAX gene (NM_002382) coding sequence, an additional two nucleotides adjacent to each exon, and 1 kb of upstream sequence. Amplicon size ranged from 125 to 275 bp (including primers) with an average of 243 bp. Inserts ranged in size from 77 to 230 bp (excluding primers), with an average of 194 bp. Ion AmpliSeq detection for homozygous deletions was performed after normalization to non-neoplastic DNA sequences and establishing cutoffs based on estimated presence of 30% non-neoplastic cells in low-/intermediate-risk GISTs and 20% non-neoplastic cells in high risk/metastatic GISTs. Deletion of at least nine consecutive amplicons and/or a ratio of <0.4 for markers located at either the 3′- or 5′-end of the gene in relation to all markers in a given case were defined as criteria for homozygous deletion.

Fastq files from the sequencer were redirected to a custom pipeline for HaloPlex^™ Target Enrichment System on the DNA nexus platform and/or to Agilent Surecall software.
Briefly, reads were aligned to the human reference genome (GRCh37/hg19) (http://hgdownload.cse.ucsc.edu/) with Burrows-Wheeler Aligner (BWA) [28 (link)] and variants were called using at least 2 of the 3 following variant callers: Genome Analysis Toolkit (GATK) [29 (link)–31 ], Freebayes [32 ] (both within the DNA nexus platform), and Base Alignment Quality (BAQ) Single Nucleotide Polymorphism (SNP) caller (within SureCall tool).
Variants passing quality filters were annotated separately against NCBI RefGene (http://www.ncbi.nlm.nih.gov) and ENSEMBL Variant Effect Predictor ver.72 (http://www.ensembl.org/info/docs/tools/vep).