Single-molecule DNA sequencing of widely varying GC-content using nucleotide release, capture and detection in microdroplets

作者：Tim J. Puchtler, Kerr Johnson, Rebecca N Palmer, Emma L. Talbot, Lindsey A Ibbotson, Paulina Klaudyna Powalowska, Rachel Knox, Aya Shibahara, Pedro Cunha, Oliver J Newell, Mei Wu, J. K. Chana, Evangelia‐Nefeli Athanasopoulou, Andreas M Waeber, Magdalena Stolarek, Ana-Luisa Silva, Justyna M Mordaka, Michael Haggis-Powell, Christina Xyrafaki, James Bush, Ibrahim S Topkaya, Maciej Sosna, Richard J. Ingham, Thomas Huckvale, Aurel Negrea, Boris Breiner, Justinas Šlikas, Douglas J. Kelly, Alexander J. Dunning, Neil M. Bell, Mark Dethlefsen, David M. Love, Paul H. Dear, Jekaterina Kuleshova, Gareth J Podd, Tom H Isaac, Barnaby W Balmforth, Cameron A Frayling · 发表于：Nucleic Acids Research · 年份：2020 · DOI：10.1093/nar/gkaa987 · 被引用次数：6 · 研究领域：Advanced biosensing and bioanalysis techniques、Protist diversity and phylogeny、Single-cell and spatial transcriptomics

Despite remarkable progress in DNA sequencing technologies there remains a trade-off between short-read platforms, having limited ability to sequence homopolymers, repeated motifs or long-range structural variation, and long-read platforms, which tend to have lower accuracy and/or throughput. Moreover, current methods do not allow direct readout of epigenetic modifications from a single read. With the aim of addressing these limitations, we have developed an optical electrowetting sequencing platform that uses step-wise nucleotide triphosphate (dNTP) release, capture and detection in microdroplets from single DNA molecules. Each microdroplet serves as a reaction vessel that identifies an individual dNTP based on a robust fluorescence signal, with the detection chemistry extended to enable detection of 5-methylcytosine. Our platform uses small reagent volumes and inexpensive equipment, paving the way to cost-effective single-molecule DNA sequencing, capable of handling widely varying GC-bias, and demonstrating direct detection of epigenetic modifications.