To investigate mutational pattern of different liver metastases, we examined biomarkers (single-nucleotide polymorphism, SNP) through genome-wide exploration using NGS. To preliminarily select genes to construct a custom panel for target capture sequencing, we performed WES for 10 triplets, each comprising primary colorectal tumor and normal colorectal mucosa and matched liver metastases. Genomic DNA from fresh tissue was samples sequenced on an Ion™ Proton (Life Technologies, Carlsbad, CA, USA) platform according to the manufacturer’s instructions. Normal colorectal mucosa was sequenced to exclude germline variants. The read alignments and variant analyses were performed according to the predefined workflow.
We constructed a custom panel of 96 genes selected based on driver mutations identified using WES and Tumor Mutation Hotspots Panel version 2 (Life Technologies, Carlsbad, CA, USA). Genomic DNA from FFPE tissue samples of patients in both cohorts was subsequently sequenced for SNPs using an Ion™ Torrent Personal Genome Machine (PGM) according to the manufacturer’s instructions. For a given gene loci, the fraction of mutant alleles was calculated by diving the number of mutant reads by the number of total reads. A 5% cutoff value was employed. A sample was considered wild-type for a given gene when all sequenced loci harbored <5% mutant alleles.
We constructed a custom panel of 96 genes selected based on driver mutations identified using WES and Tumor Mutation Hotspots Panel version 2 (Life Technologies, Carlsbad, CA, USA). Genomic DNA from FFPE tissue samples of patients in both cohorts was subsequently sequenced for SNPs using an Ion™ Torrent Personal Genome Machine (PGM) according to the manufacturer’s instructions. For a given gene loci, the fraction of mutant alleles was calculated by diving the number of mutant reads by the number of total reads. A 5% cutoff value was employed. A sample was considered wild-type for a given gene when all sequenced loci harbored <5% mutant alleles.