Exome Sequencing of Circulating Cell-Free DNA

For plasma samples, ~1 ng of cfDNA was used as the input for library preparation, and libraries were prepared using a ThruPLEX DNA-seq Library Prep Kit according to the manufacturer’s protocol. For blood samples, libraries were prepared with 200 ng of genomic DNA using a SureSelect Library Preparation Kit (Agilent Technologies) according to the manufacturer’s instructions [21 (link),55 (link)]. For exome enrichment, SureSelect Human All Exon V6 + UTRs probe sets were used. The captured libraries were amplified with 14 cycles of PCR, and the quality and concentration of libraries were assessed using the TapeStation 2200 system (Supplementary Figure S1B). Libraries were indexed with barcodes to allow sample pooling for multiplexed exome capture and sequencing. An IlluminaNovaSeq 6000 DNA sequencer (Illumina) was used to conduct WES [18 (link)].