Single-molecule DNA sequencing of widely varying GC-content using nucleotide release, capture and detection in microdroplets. Issue 22 (5th November 2020)

• Record Type: Journal Article
• Authors: Puchtler, Tim J; Johnson, Kerr; Palmer, Rebecca N; Talbot, Emma L; Ibbotson, Lindsey A; Powalowska, Paulina K; Knox, Rachel; Shibahara, Aya; M. S. Cunha, Pedro; Newell, Oliver J; Wu, Mei; Chana, Jasmin; Athanasopoulou, Evangelia-Nefeli; Waeber, Andreas M; Stolarek, Magdalena; Silva, Ana-Luisa; Mordaka, Justyna M; Haggis-Powell, Michael; Xyrafaki, Christina; Bush, James; Topkaya, Ibrahim S; Sosna, Maciej; Ingham, Richard J; Huckvale, Thomas; Negrea, Aurel; Breiner, Boris; Šlikas, Justinas; Kelly, Douglas J; Dunning, Alexander J; Bell, Neil M; Dethlefsen, Mark; Love, David M; Dear, Paul H; Kuleshova, Jekaterina; Podd, Gareth J; Isaac, Tom H; Balmforth, Barnaby W; Frayling, Cameron A;
• Is Part Of: Nucleic acids research. Volume 48:Issue 22(2020)
• Page Start: e132
• Page End: e132
• Publication Date: 2020-11-05
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkaa987
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: 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.