Sequencing data was analyzed using Torrent Suite V4.0 software and Variant Caller plug-in. Variants were evaluated using Ingenuity Variant Analysis (IVA) tool. Mutant allele frequency cut-off was set to 1%. Systematic filtering steps were incorporated to accurately identify true somatic variants. Germline variants were removed from matched cfDNA-buffy gDNA pairs within individual patient samples. CRAVAT 4 (Cancer-Related Analysis of Variants Toolkit) was used to annotate remaining variants. Further, variants occurring in 1000Genomes were removed. Additional filtering was performed to eliminate errors due to strand-bias and preserve the protein-coding and non-synonymous variants. Finally, variants were visually inspected using Integrative Genomics Viewer (IGV) software.
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