System and method for generation and use of optimal nucleotide flow orders
An embodiment of a method for generating a flow order that minimizes the accumulation of phasic synchrony error in sequence data is described that comprises the steps of: (a) generating a plurality of sequential orderings of nucleotides species comprising a k-base length, wherein the sequential orderings define a sequence of introduction of nucleotide species into a sequencing by synthesis reaction environment; (b) simulating acquisition of sequence data from one or more reference genomes using the sequential orderings, wherein the sequence data comprises an accumulation of phasic synchrony error; and (c) selecting one or more of the sequential orderings using a read length parameter and an extension rate parameter.
1. A method for sequencing a nucleic acid template using a flow order that minimizes the accumulation of phasic synchrony error in sequence data, comprising the steps of:
(a) introducing a numerically generated sequential ordering of nucleotide species comprising a k-base length into a sequencing by synthesis reaction environment, wherein the numerically generated sequential ordering of nucleotide species comprises a predetermined series of flows of nucleotide species that is generated by
(i) simulating expected flowgram signals that would be generated using a sequencing by synthesis method to acquire sequence data from one or more known reference sequences, wherein the sequence data comprises an accumulation of phasic synchrony error from the one or more known reference sequences and
(ii) selecting the series of flows of nucleotide species that demonstrates a high read length characteristic and a low extension rate characteristic for the one or more known reference sequences, wherein the high read length characteristic is greater than about 400 bp and the low extension rate characteristic is less than or equal to 0.55 bp/flow;
(b) acquiring signals from the sequencing by synthesis reaction environment in response to incorporation of the nucleotide species in an extension reaction of one or more populations of substantially identical nucleic acid template molecules, wherein the signals comprise a measure of error from a subset of nucleic acid template molecules from one or more of the populations fall behind a phase of extension; and
(c) cyclically repeating the introduction of the numerically generated sequential ordering of nucleotide species and acquisition of signals for a number of iterations, wherein the subset of nucleic acid molecules re-synchronize with the phase of extension that reduces the measure of error due to the high read length characteristic and the low extension rate characteristic of the sequential ordering.
2. The method of claim 1 , wherein:
the sequencing by synthesis reaction environment comprises an array of wells.
3. The method of claim 1 , wherein:
the k-base length is selected from the group consisting of 16, 24, 32, and 40 base lengths.
4. The method of claim 1 , wherein:
the k-base length comprises a length in a range of 32-40 bases.
5. The method of claim 1 , wherein:
the read length characteristic comprises a measure of read length that comprises less than 3% of the accumulated phasic synchrony error.
6. The method of claim 1 , wherein:
the extension rate characteristic comprises an average number of complementary sequence positions to the template molecule a single nucleotide flow can extend.
7. The method of claim 1 , wherein:
the read length characteristic is greater than about 400 bp and the extension rate characteristic is less than or equal to about 0.55 bp/flow at a 0.5% incompletion rate and a 0.5% carry-forward rate.