Avenio ctdna analysis software

The CAPP-Seq of ctDNA (10−50 ng) was performed using the AVENIO ctDNA surveillance kit targeting 197 genes (Roche Diagnostics) as previously described [26 (link),27 (link),28 (link)]. The purified libraries were pooled and sequenced on Illumina NextSeq 500 (Illumina, San Diego, CA, USA) using 300-cycle high output kit. Variants were called with the AVENIO ctDNA Analysis Software (Roche Diagnostics), which includes bioinformatics methods from CAPP-Seq [18 (link)] and integrated digital error suppression [19 (link)]. Genetic variants previously cataloged by the Exome Aggregation Consortium at a frequency of ≥1% were excluded, and only non-synonymous single nucleotide variants (SNVs), insertions–deletions (Indels), copy number variations (CNVs), and gene fusions involving 197 cancer-related genes were extracted. The blood tumor mutational burden (bTMB) in each sample was evaluated as the number of non-synonymous mutations number per Mb.

CAPP-Seq was performed with ctDNA isolated from plasma samples as previously reported^{16 (link)}. In brief, we used a maximum of 50 ng of DNA for the CAPP-Seq ctDNA analyses, which were performed using the AVENIO ctDNA surveillance kit for 197 genes (Roche Diagnostics, Indianapolis, IN), according to the manufacturer’s instructions. The AVENIO ctDNA Surveillance Kit contains major mutated genes in gynecological cancer (TP53, BRCA1, BRCA2, BRAF, PIK3CA, KRAS, NRAS, APC, EGFR, MET, ERBB2, CTNNB1, etc.). The purified libraries were pooled and sequenced on an Illumina NextSeq. 500 (Illumina, San Diego, CA) using a 300-cycle high-output kit. Variants were called with the AVENIO ctDNA analysis software (Roche Diagnostics), which includes bioinformatics methods from CAPP-Seq^{15 (link)} and integrated digital error suppression^{16 (link)}. Germline mutations were excluded based on the Human Genetic Variation Database (http://www.genome.med.kyoto-u.ac.jp/SnpDB) and the Exome Aggregation Consortium database^{33 (link)}.

ctDNA data used in this study were generated using capture-based targeted sequencing and shallow whole genome sequencing (sWGS), as previously reported [16 (link), 17 (link)]. Briefly, sequencing libraries were prepared using the AVENIO ctDNA Library Preparation Kit with either the Targeted or Surveillance Panel (Roche Diagnostics). Library pools were sequenced on the Illumina NextSeq 550 platform with the High-Output Kit V2 (2 × 150 bp). Downstream analysis was performed using the AVENIO ctDNA analysis software (Roche Diagnostics, version 2.0.0), applying a variant allele frequency threshold of 0.01%. In parallel, libraries for sWGS were prepared using the KAPA HyperPrep Kit with KAPA Dual-Indexed Adaptors, and sequenced on the Illumina HiSeq 4000 platform (2 × 100 bp). Genome-wide copy number profiles were estimated using ichorCNA [21 (link)]. Trimmed Median Absolute Deviation from copy number neutrality (t-MAD) scores were calculated as previously described [17 (link), 18 (link)].

We analyzed the mutation profile and TMB as previous study^{11 (link)}; A maximum of 50 ng of DNA was used for the CAPP-Seq ctDNA analyses using the AVENIO ctDNA surveillance kit (Roche Diagnostics, 197 genes). The purified libraries were pooled and sequenced on an Illumina NextSeq. 500 sequencing system (Illumina) using the 300-cycle high output kit. Variants were called with the AVENIO ctDNA Analysis Software (Roche Diagnostics), which includes bioinformatics methods from CAPP-Seq2^{12 (link)} and integrated digital error suppression^{13 (link)}. Germline mutations were excluded with the use of the Human Genetic Variation Database (http://www.genome.med.kyoto-u.ac.jp/SnpDB) and the ExAC database.

Following sequencing, alignment and gene variant calling were performed using the AVENIO ctDNA analysis software (Roche Diagnostics), with default parameter settings for the expanded panel. The analysis software includes three default reports that are automatically generated: a sample metrics report, an initial variant report (unfiltered or all variants), and a second variant report (Roche default filter). The percentage of aligned reads to the human genome that are within the targeted region (unique depth) according to the manufacturer’s instructions should be >40%. Similarly, the expected median unique depth across bases in the targeted region should be at least 2500×, given 50 ng input cfDNA.
All variants were manually inspected and gene variants present in population databases (EXAC, dbSNP, 1000 genomes) were not considered as relevant. To investigate pathogenicity value, the target variants were submitted to the disease-associated databases COSMIC, VARSOME, and Onco KB, and only variants annotated as pathogenic or likely pathogenic were taken into account.
The level of actionability of specific alterations was evaluated with the ESMO Scale of clinical actionability of molecular targets (ESCAT) [11 (link)].

CfDNA was quantified using the Qubit dsDNA High Sensitivity Kit (Life Technologies). The median DNA input for library preparation was 50 ng. Libraries were prepared with the AVENIO ctDNA Expanded Kit (Roche Sequencing Solutions, Pleasanton, CA, USA) according to the manufacturer’s instructions. Multiplex libraries consisting of 10 unique samples were sequenced on a NextSeq 500 High Output lane (Illumina, San Diego, CA, USA) using 150 bp paired-end runs (median unique depth 4524×, range 1420–9664). Sequencing data were processed using the AVENIO ctDNA Analysis Software version 1.1 with the Expanded Panel Workflow (Roche). Variants were accepted if they were previously reported to the Catalogue of Somatic Mutations in Cancer (COSMIC) or the Cancer Genome Atlas (TCGA). Variants with a mutant AF > 0.1% in any of the Exome Aggregation Consortium (ExAC), 1000 Genomes Project, and Single Nucleotide Polymorphism (dbSNP) databases were excluded. To ensure specificity, variants were required to be present in 3 unique reads and have AF > 0.1% to be accepted [38 (link)].