Targeted DNA Sequencing for Mutational Profiling

DNA was extracted from FFPE primary tumor biopsies using a QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). DNA quality was evaluated based on the absorbance ratios of A260/280 and A260/230 using a NanoDropTM 2000c Spectrophotometer (Thermo Fisher, MA, USA). DNA quantity was determined using the Qubit^® 2.0 Fluorometer with the Qubit^® dsDNA HS Assay Kit (Thermo Fisher). Two independent Targeted DNA sequencing panels were employed to allow the mutational profiling of 72 cancer driver genes (see Supplementary Data 1). DNA libraries that were built with GeneRead DNAseq Colorectal Cancer Panel V2 were processed and analyzed as was previously described (31 (link)). Of note, DNA libraries that were constructed with the AmpliSeqTM for Illumina Cancer Hotspot Panel v2 Kit that allow the detection of 2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes, were prepared with 100 ng of genomic DNA as was previously described (40 (link)). These DNA libraries were measured using Qubit^® 2.0 Fluorometer with the Qubit^® dsDNA HS Assay Kit (Thermo Fisher). All libraries were above the minimum concentration requirement of 2 nM for further sequencing in an Illumina MiSeq platform.