(a) Raw images averaged from selected individual flows. Example images for each of the nucleotides from indicated flow numbers were averaged from the time lapsed frame numbers, 50 to 150, which corresponds to 10 s of data. These frame numbers capture the rise, peak and fall of sequencing chemistry. (b) From a ROI in the FOV for a synthetic template, ~357 time traces were extracted and background corrected from each DNA cluster and for each individual flow. The 357 time traces were averaged and colour coded to indicate the correct sequence. Numbers are given as reference points for indicating flow numbers and numbers in parentheses indicate expected homopolymer repeats for the given flow. Flow order was arbitrarily chosen as ‘G’, ‘C’, ‘A’ and ‘T’. Correct base calls are indicated in capitalized coloured text and homopolymer repeats are indicated by subscripts, and negative flows are indicated by lower case text. (c) Averaged time traces for flows 3, 9, 18, 27 and 44, which correspond to negative, G, C, A and T 1-mer flows, respectively.