Targeted bisulfite sequencing for adjacent normal tissue
DNA libraries were prepared from bisulfite-converted gDNA and cfDNA using the Hieff NGS Methyl-seq DNA Library Prep Kit for Illumina (Yeasen, Shanghai, China) and an in-house developed kit, respectively. The concentration and size of the libraries were detected using the QubitTM dsDNA HS Assay Kit (Invitrogen, USA) and D1000 Reagents (Agilent, USA), respectively. The libraries were subsequently hybridized to the Twist Human Methylome Panel (Twist Biosciences, South San Francisco, CA, USA), which covers 3.98 M of CpG sites through 123 Mb of genomic content to target biologically relevant methylation markers. The captured libraries were then pooled at equimolar concentrations and sequenced on a NovaSeq 6000 sequencer at 150 bp paired end reads.