Avenio ctdna library preparation kit

Information on cancer-specific genomic alterations of the patient plasma samples profiled in this study was obtained from previously published work (for detailed descriptions see [2 , 4 (link)]). Somatic mutations and EML4-ALK fusion abundances were determined by hybrid-capture sequencing using the AVENIO ctDNA Library Preparation Kit followed by sequencing with the Targeted or Surveillance Panel (Roche, Mannheim, Germany). Mutations with variant allele frequencies (VAFs)≥ 30% were considered germline mutations and consequentially excluded from further analyses. VAFs < 0.01% were deemed undetectable. Genome-wide copy number profiles and t-MAD scores were estimated from sWGS data using the ichorCNA algorithm [47 (link)] and t-MAD score calculation documentation (https://github.com/sdchandra/tMAD) [29 ], respectively. CNA calling was carried out at 1-Mb bin sizes using sWGS data of 16 healthy control samples as copy number neutral references. The sequencing data was downsampled to 5 M paired reads prior to t-MAD score calculation. The maximal t-MAD score across all healthy control samples (0.0081) was set as the detection threshold. Chromosomal instabilities were similarly assessed from cfMeDIP-seq data with 5-mC-enriched sequencing data of healthy controls (n = 13) as copy number neutral reference for normalization.

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)].