title: Assessment of RainDrop BS-seq as a method for large-scale, targeted bisulfite sequencing.
abstract: We present a systematic assessment of RainDrop BS-seq, a novel method for large-scale, targeted bisulfite sequencing using microdroplet-based PCR amplification coupled with next-generation sequencing. We compared DNA methylation levels at 498 target loci (1001 PCR amplicons) in human whole blood, osteosarcoma cells and an archived tumor tissue sample. We assessed the ability of RainDrop BS-seq to accurately measure DNA methylation over a range of DNA quantities (from 10 to 1500 ng), both with and without whole-genome amplification (WGA) following bisulfite conversion. DNA methylation profiles generated using at least 100 ng correlated well (median R = 0.92) with those generated on Illumina Infinium HumanMethylation450 BeadChips, currently the platform of choice for epigenome-wide association studies (EWAS). WGA allowed for testing of samples with a starting DNA amount of 10 and 50 ng, although a reduced correlation was observed (median R = 0.79). We conclude that RainDrop BS-seq is suitable for measuring DNA methylation levels using nanogram quantities of DNA, and can be used to study candidate epigenetic biomarker loci in an accurate and high-throughput manner, paving the way for its application to routine clinical diagnostics.
authors: UCL Cancer Institute; University College London; London, United Kingdom.
authors: Dirk S
authors: DS
authors: Paul
authors: UCL Cancer Institute; University College London; London, United Kingdom.
authors: Paul
authors: P
authors: Guilhamon
authors: UCL Cancer Institute; University College London; London, United Kingdom.
authors: Anna
authors: A
authors: Karpathakis
authors: UCL Cancer Institute; University College London; London, United Kingdom.
authors: Lee M
authors: LM
authors: Butcher
authors: UCL Cancer Institute; University College London; London, United Kingdom.
authors: Christina
authors: C
authors: Thirlwell
authors: UCL Cancer Institute; University College London; London, United Kingdom.
authors: Andrew
authors: A
authors: Feber
authors: UCL Cancer Institute; University College London; London, United Kingdom.
authors: Stephan
authors: S
authors: Beck
mesh_terms: D001859:Bone Neoplasms / Q000235:genetics; D018278:Carcinoma, Neuroendocrine / Q000235:genetics / Q000556:secondary; D018899:CpG Islands; D019175:DNA Methylation; D006801:Humans; D007202:Indicators and Reagents; D009852:Omentum / Q000473:pathology; D012516:Osteosarcoma / Q000235:genetics; D010534:Peritoneal Neoplasms / Q000235:genetics / Q000556:secondary; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000737:chemistry*; D014407:Tumor Cells, Cultured
chemical_list: D007202:Indicators and Reagents; D013447:Sulfites; C042345:hydrogen sulfite
keywords: DNA methylation; EWAS; RainDrop BS-seq; epigenetic biomarker; microdroplet PCR; targeted bisulfite sequencing