Capture-based targeted sequencing of ccfDNA
AMPLIFICATION, SENSITIVITY
Target enrichment was performed according to the xGen hybridization capture of DNA libraries protocol (Integrated DNA Technologies, Inc). In brief, equimolar amounts of six different libraries with unique indices were pooled (3 µg total library input) in a single capture hybridization reaction, and human Cot DNA and xGen Universal Blockers-TS Mix (both Integrated DNA Technologies, Inc) were added as blocking agents. Our CheCUP panel probes were hybridized to the libraries using the xGen Hybridization and Wash Kit (Integrated DNA Technologies, Inc) for 16 h at 65 °C. The capture reaction and extensive washing steps were performed using Dynabeads M-270 Streptavidin beads and xGen Hybridization and Wash Kit, respectively, according to the manufacturer’s protocol. Captured libraries were on-beads amplified in 16 PCR cycles with xGen Library Amplification Primers (Integrated DNA Technologies, Inc) complementary to the P5 and P7 sequences of Illumina adapters and KAPA HiFi HotStart ReadyMix (Roche Diagnostics International AG). The post-capture libraries were purified using SPRIselect (Beckman Coulter) and qPCR-based quantified with adapter-specific primers using the KAPA Library Quantification Kit (Roche Diagnostics International AG). Measurement of average fragment length was performed with TapeStation 4150 using the High Sensitivity D1000 ScreenTape Assay (Agilent Technologies) according to the manufacturer’s instructions. Always 54 equimolar multiplexed target-enriched libraries (10 nM multiplex input) were then sequenced using 150 bp paired-end runs on Illumina NovaSeq 6000 S4 v.15 NGS platform (Illumina) at the DKFZ NGS Core Facility according to the manufacturer’s instructions. The quality scores Q30 were between 90% and 89%, total read count was 44 937 007x mean coverage per library (range: 7 347 738x – 153 278 584x). We achieved 424 535x mean target coverage (median: 412 567x; range: 30 792x – 1 000 042x) prior duplication removal and 1 678x average unique target coverage (median: 1 607x; range: 262x – 4 376x) after deduplication.