CtDNA Sequencing and Copy Number Profiling

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)].