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

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.

Comparative Analyses of Targeted Myeloid Cancer Next-Generation Sequencing Panel in Fresh Blood, Bone Marrow and FFPE Material

“…This sequencing technique does not rely on PCR, as DNA or RNA can be sequenced directly. Therefore, this single-molecule DNA sequencing seems to be an efficient method for sequencing GC or repeat-rich regions and appears to be a suitable addition to established NGS applications [ 41 , 42 ].…”

A programmable and automated optical electrowetting-on-dielectric (oEWOD) driven platform for massively parallel and sequential processing of single cell assay operations

“…In both oEWOD and EWOD the droplets are manipulated using a regional change in the contact angle between the droplets and the surface. 20 In oEWOD the electrode shape is varied by using localised illumination to alter the conductivity of a photoactive layer built into the chip surface. This removes the need for complex pre-fabricated electrode arrays.…”

A programmable and automated optical electrowetting-on-dielectric (oEWOD) driven platform for massively parallel and sequential processing of single cell assay operations

“…In both oEWOD and EWOD the droplets are manipulated using a regional change in the contact angle between the droplets and the surface . 20 In oEWOD the electrode shape is varied by using localised illumination to alter the conductivity of a photoactive layer built into the chip surface. oEWOD has previously been described as a practical and broadly applicable method of droplet manipulation allowing the versatility of EWOD whilst requiring relatively simple device fabrication.…”