Targeted ctDNA Sequencing for Cancer

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.