Avenio ctdna targeted kit

For mutation profiling, we used the commercially available AVENIO ctDNA Targeted Kit, which covers 17 genes across 81 kb, including those in the U.S. National Comprehensive Cancer Network (NCCN) Guidelines, using hybrid capture targeted enrichment techniques (ALK, APC, BRAF, BRCA1, BRCA2, DPYD, EGFR, ERBB2, KIT, KRAS, MET, NRAS, PDGFRA, RET, ROS1, TP53, UGT1A1) (Roche). In brief, sequencing libraries were generated from 10–20 ng of plasma-derived DNA according to the manufacturer’s instructions. Libraries were quantified by qPCR and the quality was assessed using a Bioanalyzer High Sensitivity Kit (Agilent Technologies). Pooled libraries were sequenced on the Illumina NextSeq platform using a 2x150bp paired-end mode. On average, 15 million paired-end reads were obtained per sample (range 12.7–17.3 M). Sequencing data were analyzed using the AVENIO ctDNA Analysis Software (Roche) and variant calls were generated with a customized workflow using defined somatic variant filter settings. We excluded synonymous variants and variants present with >1% mutant allele frequency in population frequency databases (ExAC, gnomAD, 1000genomes).

Costs for cfDNA analysis were based on a recent microcosting analysis.(unpublished) The microcosting analysis was an activity-based costing method, and includes the costs for personnel, materials, and equipment. Personnel costs include costs for all staff (i.e., technicians, pathologists, and clinical molecular pathologists). Several assumptions were made to calculate the cost price. The cost price was based on analyzing 12 samples per week in one run, assuming that not all runs will be completely full. In line with the costing of tissue-based diagnostics, a 44% markup was used to account for overhead [14 ]. We assumed that these covered the costs for multipurpose equipment and all other indirect costs. A 0.5% failure rate was included in the costs [11 (link)]. Utilization of equipment was assumed to be 33%. Steps of the microcosting framework include sample collection, internal sample transport, sample processing, cfDNA isolation with QiaSymphony SP (Qiagen, Germantown, MD, USA), ctDNA analysis with Avenio ctDNA Targeted Kit (Roche Diagnostics, Pleasanton, CA, USA) + NextSeq 550 (Illumina, San Diego, CA, USA), and reporting of results. Costs are listed in supplementary Table S2. Costs for material and equipment include 21% VAT.