Raw data associated with the article: "Single-molecule DNA sequencing of widely varying GC-content using nucleotide release, capture and detection in microdroplets.", NAR, Puchtler et.al.

All data taken in the production of the corresponding paper: "Single-molecule DNA sequencing of widely varying GC-content using nucleotide release, capture and detection in microdroplets."

The associated manuscript describes a method for DNA sequencing which involves the sequential release of nucleotides from a single, immobilised strand of DNA via pyrophosphorolysis (PPL). Released nucleotides, in the form of dNTPs, are captured in microdroplets which are manipulated using an optical-EWOD platform. A detection chemistry within each droplet releases a specific dye depending on which dNTPs are present, allowing the optical read-out of bases within each droplet. Hence, by capturing bases sequentially within droplets as they are cleaved from the strand of DNA, the sequence can be optically identified.

Methods
Manipulation of the droplets, each containing the required reagents for PPL, is performed using a photoactive-dielectric chip for optical electrowetting-on-dielectric (oEWOD). This chip is addressed using programmable illumination from a DMD-projector setup, with droplets in oil between the dielectric stack.

DNA is immobilised on the sample surface by binding to a silica microsphere and subsequentially trapping the bead using compression.

Once droplets containing the reagents necessary for PPL are passed over the DNA, they are mixed with the detection reagents and kept in-order. They are subsequentially scanned using a standard fluorescence microscope, where each wavelength band used corresponds to a difference base present.

All data is derrived from fluorescence images of the droplets after they have captured the released nucleotides, with the parameters of droplet size and brightness in each channel being recorded (see 'Basecalling' tabs in data). It is from this intensity data per detection channel that the presence of nucleotides can be determined, and hence the sequence of released nucleotides reconstructed, allowing a sequence to be generated.

All methods for collection of this data are presented in detail in the corresponding manuscript.