DOI: 10.5061/dryad.4xgxd2575
title: 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.

description: 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.
description: 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.
description: Whilst the majority of data presented is self-explanatory with
 the associated manuscript, some notes should be provided for the
 'Basecalling' tabs, as this is more complicated:<br> -
 Each row represents a different identified droplet taken from the
 fluorescence scan.<br> - Of these, entries with 'RA
 Success' should be taken, denoting that the droplets have
 successfully been merged (PPL and DET mixes) after passing over DNA, as
 determined by background fluorescence in a tracer channel (not linked to
 dNTP detection fluors)<br> - For these droplets, the raw data is
 essentially "size" and intensities in 4 scanning channels (i.e.
 "I_532", "I594", "I_633" and
 "I_700"). This is the data presented in the manuscript
 figures. <br> - These intensities are plotted, as in the manuscript,
 and the distributions used to associate occupation of the bases. The
 decision of whether each droplet belongs to the 'occupied' or
 'unoccupied' peaks is written in the columns labelled
 "called_532", "called_594" etc. It is from this that
 sequence data is generated.<br> - All other data in the
 'Basecalling' tabs are additional estimations of the liklihood
 of misassigning the droplets to the wrong catagory, or error handeling for
 other models used. They may be of interest, but are not in any way key to
 the dataset as already described. One other set of data
 should be given additional user notes, "False-Signal Analysis".
 In this, blocks of data are grouped vertically for a given sequence of
 DNA, and horisontal groupings are for every identified droplet within an
 experiment ("All droplets"), those which have not passed over
 DNA but still contain PPL and detection reagents (denoted
 "Merge-only") and those which have actually passed over the DNA
 and are to be used for sequencing ("DOB-1s). Each block contains the
 data of one experiment, and data for each scan wavelength measured.
 Estimations are then made for the likely concentration of dNTPs present
 ("scan concentration") based off estimations of the size of
 droplets and their intensities, as well as the overlap between
 'occupied' and 'unoccupied' peaks of the intensity
 distribution, as described previously ("FP" and "FN").
 Concentrations of dNTPs are also put into a 'per droplet' value
 ("bases per drop").