Single-stranded pre-methylated 5mC adapters uncover the methylation profile of plasma ultrashort Single-stranded cell-free DNA

生物 DNA甲基化 DNA 甲基化 遗传学 分子生物学 基因 基因表达
作者
Jordan Cheng,Neeti Swarup,Marco Morselli,Wei-Lun Huang,Mohammad Aziz,Christa Caggiano,Misagh Kordi,Abhijit A. Patel,David Chia,Yong Kim,Feng Li,Wei Fang,Noah Zaitlen,Kostyantyn Krysan,Steve Dubinett,Matteo Pellegrini,David T. Wong
出处
期刊:Nucleic Acids Research [Oxford University Press]
卷期号:52 (11): e50-e50 被引量:1
标识
DOI:10.1093/nar/gkae276
摘要

Abstract Whole-genome bisulfite sequencing (BS-Seq) measures cytosine methylation changes at single-base resolution and can be used to profile cell-free DNA (cfDNA). In plasma, ultrashort single-stranded cfDNA (uscfDNA, ∼50 nt) has been identified together with 167 bp double-stranded mononucleosomal cell-free DNA (mncfDNA). However, the methylation profile of uscfDNA has not been described. Conventional BS-Seq workflows may not be helpful because bisulfite conversion degrades larger DNA into smaller fragments, leading to erroneous categorization as uscfDNA. We describe the ‘5mCAdpBS-Seq’ workflow in which pre-methylated 5mC (5-methylcytosine) single-stranded adapters are ligated to heat-denatured cfDNA before bisulfite conversion. This method retains only DNA fragments that are unaltered by bisulfite treatment, resulting in less biased uscfDNA methylation analysis. Using 5mCAdpBS-Seq, uscfDNA had lower levels of DNA methylation (∼15%) compared to mncfDNA and was enriched in promoters and CpG islands. Hypomethylated uscfDNA fragments were enriched in upstream transcription start sites (TSSs), and the intensity of enrichment was correlated with expressed genes of hemopoietic cells. Using tissue-of-origin deconvolution, we inferred that uscfDNA is derived primarily from eosinophils, neutrophils, and monocytes. As proof-of-principle, we show that characteristics of the methylation profile of uscfDNA can distinguish non-small cell lung carcinoma from non-cancer samples. The 5mCAdpBS-Seq workflow is recommended for any cfDNA methylation-based investigations.
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