Single-stranded Premethylated 5mC Adapters Uncovers the Methylation Profile of Plasma Ultrashort Single-Stranded Cell-Free DNA

We describe an optimized library preparation protocol for uscfDNA in which single stranded 5 methylcyotosine premethylated adapters are attached to cell-free DNA fragments prior to bisulfite conversion and sequencing to assess methylation patterns. This method improves accuracy of downstream uscfDNA analysis by prevented bisulfite conversion degraded DNA from being incorporated into the final library. Using this technique, we report that bisulfite-converted uscfDNA and mncfDNA map at different genomic positions and that uscfDNA CpG methylation is globally lower than mncfDNA by 20 percent. As a proof of concept, we show that the methylation characteristics of uscfDNA can be used to distinguish NSCLC and non-cancer subjects through differentiated methylated region analysis and other epigenetic patterns.