ccfDNA isolation and sequencing library preparation
SENSITIVITY
ccfDNA from healthy control samples and CUP patients was isolated from plasma (median: 10.2 ml; range: 4.3–14.2 ml) using the QIAamp MinElute ccfDNA Midi Kit (Qiagen) following the manufacturer’s instructions and stored at −80 °C. Purified DNA was quantified with Qubit 2.0 using the dsDNA High Sensitivity Kit (Thermo Fisher Scientific). Size distribution of isolated DNA was analyzed with TapeStation 4150 using the cell-free DNA ScreenTape Assay (Agilent Technologies) according to the manufacturer’s instructions. For both capture-based targeted and shallow whole-genome sequencing, between 8 and 40 ng of ccfDNA were size-selected for DNA fragments between 90 and 220 bp using SPRIselect (Beckman Coulter) according to the manufacturer’s recommendations.Sequencing libraries were prepared using the KAPA Hyper Prep Kit (Roche Diagnostics International AG) with an optimized manufacturer’s protocol and without additional DNA fragmentation. In brief, up to 4 ng (median: 4 ng; range: 1.4–4 ng) of size-selected ccfDNA were subjected to end-repairing, A-tailing and adapter ligation with xGen Dual Index UMI adapters (Integrated DNA Technologies, Inc). These unique adapters enable superior low-frequency variant detection in low-input samples like ccfDNA and contain both unique molecular identifiers (UMIs) barcoding individual molecules in a sample and unique combined P5 and P7 dual-index sequences that are both unique to a single sample. Following adapter ligation for 15 h at 16 °C to achieve high ligation efficiency, libraries were purified using SPRIselect (Beckman Coulter) according to the manufacturer’s protocol. Finally, libraries were amplified in 11-12 PCR cycles and the clean-up of amplified libraries were performed according to the manufacturer’s protocol. The size distribution of libraries was confirmed with TapeStation 4150 using the High Sensitivity D1000 ScreenTape Assay (Agilent Technologies) according to the manufacturer’s instructions. Finally, the amplified libraries were qPCR-based quantified with adapter-specific primers using the KAPA Library Quantification Kit (Roche Diagnostics International AG) and utilized for both capture-based targeted sequencing and shallow whole-genome sequencing as detailed below.