Targeted sequencing

Targeted capture-based sequencing of pretreatment (baseline) and progression plasma DNA was performed using the Avenio ctDNA Analysis Expanded Kit (Roche diagnostics) following manufacturer's protocols. On the basis of the previously published cancer, personalized profiling by deep-sequencing (CAPP-seq) methodology, this covers a pan cancer panel of 77 genes optimized for use in colorectal cancer and NSCLC (34). Between 6 and 10 ng of genomic DNA were used for library construction and the purified libraries were pooled and sequenced on an Illumina NextSeq 500 (illumina). Variants were called using a specialized bioinformatic analysis workflow, which uses integrated digital error suppression (iDES) system that augments CAPP-seq through in silico removal of stereotypical sequencing artifacts combined with molecular barcoding (35). Only nonsynonymous single nucleotide variants (SNV), insertions-deletions (Indels), copy-number variations (CNV), and gene fusions were extracted for analysis.