Fig. 3. De novo sequencing accuracy of ALIEN DNA.

A Three representative variable voltage reads for the ALIEN DNA section of Test sequence 1. Recurring features are labelled by Greek letters. B The variable voltage conductance curve prediction for the true sequence of Test sequence 1 with the same features labelled by Greek letters. Individual de novo base calls for each read are shown below aligned to the true sequence. Because the 4-mer map does not include mixtures of ACGT and PZSB DNA, Sequencing accuracy is only evaluated for the central 24 nucleotides of the 28-base ALIEN DNA region. Thus base-call accuracies are an estimate of bulk sequencing accuracy without edge effects. Sequences displayed are in time-order so appear 3′ to 5′ because Hel308 helicase walks from 3′ to 5′. C Single-read variable-voltage sequencing accuracy and (D) basecall confusion matrix of 4 pseudorandom strands of ALIEN DNA using the k-mer model. We find that B and S are miscalled more often than P and Z. Average per-base sequencing accuracy is 74 ± 1% (s.e.m.) for single passages (reads) of single DNA molecules, substantially better than random accuracy (57.0 ± 0.1% s.e.m., Supplementary Fig. 9) and comparable to per-base variable-voltage sequencing accuracy of standard DNA (79.3 ± 0.3% s.e.m.). Random basecall accuracy exceeds 25% because accuracy is computed by first aligning the called sequence to the true sequence while allowing for gaps, then comparing the number of matches versus errors. Test sequences 1-4 were read 18, 21, 43, and 20 times, respectively.