Single-molecule DNA sequencing of widely varying GC-content using nucleotide release, capture and detection in microdroplets
Author:
Puchtler Tim J1ORCID, Johnson Kerr1ORCID, Palmer Rebecca N1, Talbot Emma L1ORCID, Ibbotson Lindsey A1, Powalowska Paulina K1, Knox Rachel1, Shibahara Aya1, M. S. Cunha Pedro1, Newell Oliver J1, Wu Mei1, Chana Jasmin1, Athanasopoulou Evangelia-Nefeli1, Waeber Andreas M1, Stolarek Magdalena1, Silva Ana-Luisa1, Mordaka Justyna M1, Haggis-Powell Michael1, Xyrafaki Christina1, Bush James1, Topkaya Ibrahim S1, Sosna Maciej1, Ingham Richard J1, Huckvale Thomas1, Negrea Aurel1, Breiner Boris1, Šlikas Justinas1, Kelly Douglas J1, Dunning Alexander J1, Bell Neil M1, Dethlefsen Mark1, Love David M1, Dear Paul H1, Kuleshova Jekaterina1, Podd Gareth J1, Isaac Tom H1, Balmforth Barnaby W1ORCID, Frayling Cameron A1ORCID
Affiliation:
1. Base 4 Innovation Ltd, Broers Building, JJ Thomson Avenue, Cambridge CB3 0FA, UK
Abstract
AbstractDespite 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.
Funder
Base4 Innovation, Ltd.
Publisher
Oxford University Press (OUP)
Cited by
4 articles.
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