Avenio ctdna expanded kit

The AVENIO ctDNA expanded kit (Roche Diagnostics) is a hybridisation capture sequencing-based 77 genes pan-cancer assay (online supplemental table S1). AVENIO cfDNA Isolation Kit (Roche Diagnostics) was used to extract cfDNA from plasma according to the user’s manual. The extracted cfDNA was analysed by Agilent High Sensitivity DNA Analysis Kit (Agilent Technologies, California, USA) and Qubit dsDNA HS Assay Kit (ThermoFisher Scientific, California, USA) for quality control, and then used for library preparation with 10–50 ng cfDNA input. Prepared libraries were sequenced on the NextSeq 500 500/550 High Output Kit V2 (300 cycles) on Illumina NextSeq sequencing platform (Illumina, California, USA) and analysed by the AVENIO oncology analysis software V.2.0.0 (Roche Diagnostics) according to manufacturer’s instructions.24 25 (link)

Venous blood was extracted at each timepoint and collected in STRECK Cell-Free DNA BCT tubes. Plasma was first separated from the peripheral blood cells by centrifugation at 2,800 rpm for 10 minutes at 4°C, then aliquoted in a 1.5 mL tube, and immediately stored in a deep freezer at −80°C. Cell-free circulating DNA (cfcDNA)—that is released into the peripheral blood due to apoptosis, necrosis, or active release (28 (link))—was extracted from plasma using the AVENIO cfDNA isolation kit (Roche Sequencing) and quantified by Qubit fluorometer (Thermo Fisher Scientific). Purity of cfcDNA was assessed by electrophoresis (4200 TapeStation system, Agilent) to discard the presence of genomic DNA contamination.
Libraries were prepared with approximately 20 to 50 ng of cfcDNA extracted from plasma samples using a broad targeted next-generation sequencing-based 77-gene panel (Avenio ctDNA Expanded Kit, Roche Sequencing), including coverage of the most prevalent tumor suppressor genes in human cancers (Supplementary Table S6). Libraries were sequenced in a NextSeq platform (Illumina) and the Avenio Oncology Analysis Software version 2.0.0 was used for FASTQ trimming, alignment to the reference genome, generation of variant calling files, and variant annotation.

Cell free DNA extraction from plasma and targeted sequencing was performed using the AVENIO ctDNA Expanded Kit (Roche Diagnostics), a panel of 77-gene cancer-related biomarkers. Extracted DNA was quantified using fluorimetry (Qubit, Thermo-Fisher Scientific) and stored at −20°C prior to use. Plasma cfDNA was sequenced using the Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) method for quantifying ctDNA as previously described.^{12 (link)} The method combines optimized library preparations with a multi-phase bioinformatics informatics approach to design cancer specific DNA “selectors,” that target recurrently mutated regions in the cancer of interest. Sequencing was performed on libraries generated from approximately 10 ng total cfDNA. AVENIO ctDNA Analysis Software version 2.0.0 was used to align, call, and filter variants against the hg38 human reference genome. Nonsynonymous, non-germline mutations, known to be mutated in cancer were considered significant. Buffy coat DNA was isolated and sequenced using AVENIO Tumor Tissue Expanded Kit (Roche Diagnostics) that contains the same 77-gene panel, and genes associated with clonal hematopoiesis were removed: ASXL1, PPM1D, DNMT3A, TET2, GNB1, CBL, JAK2, STAT2, MYD88, SF3B1. Both filtered and raw data were obtained.