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

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.

Single-molecule detection of deoxyribonucleoside triphosphates in microdroplets.

A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.

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

Recently, there has been an increasing emphasis on single cell profiling for high-throughput screening workflows in drug discovery and life sciences research. However, the biology underpinning these screens is often complex and is insufficiently addressed by singleplex assay screens. Traditional single cell screening technologies have created powerful sets of 'omic data that allow users to bioinformatically infer biological function, but have as of yet not empowered direct functional analysis at the level of each individual cell. Consequently, screening campaigns often require multiple secondary screens leading to laborious, time-consuming and expensive workflows in which attrition points may not be queried until late in the process. We describe a platform that harnesses droplet microfluidics and optical electrowetting-on-dielectric (oEWOD) to perform highly-controlled sequential and multiplexed single cell assays in massively parallelised workflows to enable complex cell profiling during screening. Soluble reagents or objects, such as cells or assay beads, are encapsulated into droplets of media in fluorous oil and are actively filtered based on size and optical features ensuring only desirable droplets (e.g. single cell droplets) are retained for analysis, thereby overcoming the Poisson probability distribution. Droplets are stored in an array on a temperature-controlled chip and the history of individual droplets is logged from the point of filter until completion of the workflow. On chip, droplets are subject to an automated and flexible suite of operations including the merging of sample droplets and the fluorescent acquisition of assay readouts to enable complex sequential assay workflows. To demonstrate the broad utility of the platform, we present examples of single-cell functional workflows for various applications such as antibody discovery, infectious disease, and cell and gene therapy.