Ez 96dna methylation lightning magprep

DNA was sequenced using an enhanced linear-splinter amplification sequencing (ELSA-seq) method as described previously (16 (link)). Extracted DNA was first converted to single-strand DNA molecules with sodium bisulfite (EZ-96 DNA Methylation-Lightning MagPrep, Zymo Research, Irvine, CA, USA), which was ligated to a splinted adapter in the presence of extension primers. A uracil-tolerating DNA polymerase was used to generate whole-genome bisulfite sequencing (BS-seq) libraries. Target enrichment was completed with lung cancer-specific methylation profiling RNA baits and further quantified with real-time polymerase chain reaction (PCR; Kapa Biosystems, Wilmington, MA, USA) and sequenced on a NovaSeq 6000 (Illumina, San Diego, CA, USA) using 2×150 bp cycles.
BS-seq data were further analyzed using an optimized pipeline. Raw data were trimmed using Trimmomatic (v.0.32) and then aligned using BWA-meth (v.0.2.2). After alignment, PCR duplicates were marked with Samblaster (v.0.1.20). The low mapping quality (mapping quality <20) or improper pairing reads were cleared from further analyses with Sambamba (v.0.4.7). The overlapping reads were removed by in-house scripts to avoid the double-counting of methylation signals.