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.

The emerging field of liquid biopsy stands at the forefront of novel diagnostic strategies for cancer and other diseases. Liquid biopsy allows minimally invasive molecular characterization of cancers for diagnosis, patient stratification to therapy, and longitudinal monitoring. Liquid biopsy strategies include detection and monitoring of circulating tumor cells, cell-free DNA, and extracellular vesicles. In this review, we address the current understanding and the role of existing liquid-biopsy-based modalities in cancer diagnostics and monitoring. We specifically focus on the technical and clinical challenges associated with liquid biopsy and biomarker development being addressed by the Liquid Biopsy Consortium, established through the National Cancer Institute. The Liquid Biopsy Consortium has developed new methods/assays and validated existing methods/technologies to capture and characterize tumor-derived circulating cargo, as well as addressed existing challenges and provided recommendations for advancing biomarker assays.