Predictive Model for Use in Sequencing-by-Synthesis
A method of obtaining a more accurate estimate of a signal correction parameter(s) in sequencing-by-synthesis operations, such as incomplete extension rates, carry forward rates, and/or signal droop rates. The sequencing operation produces signal data. A model is constructed to simulate a population of template strands as it undergoes the sequencing process and becomes divided into different phase-states as the sequencing-by-synthesis progresses. For example, the model may be a phase-state model. The output from the model is used to adjust the signal correction parameter(s). For example, the model may be fitted to the signal data. This fitting results in a more accurate estimate of the signal correction parameter(s). In another embodiment, the signal droop rate is modeled as a decaying function and this decaying function is fitted to the signal data to obtain an improved estimate of the signal droop rate.
1 . A method of sequencing a polynucleotide strand, comprising:
(a) receiving signal data relating to chemical reactions resulting from a flow of nucleotides;
(b) determining, using the signal data, sequence information of at least a portion of the polynucleotide strand;
(c) constructing a phase-state model for a set of flows that encompasses the sequence information, wherein the model includes a signal correction parameter and wherein the model is stored in a machine-readable memory;
(d) calculating, using the phase-state model, predicted signals resulting from the set of nucleotide flows;
(e) comparing the predicted signals to the signal data;
(f) adjusting the signal correction parameter of the phase-state model based on the comparison of the predicted signals to the signal data; and
(g) storing the adjusted signal correction parameter in the memory.
2 . The method of claim 1 , wherein the nucleotides are flowed onto an array having multiple wells, wherein the polynucleotide strand is contained in a first well of the array, and further comprising:
obtaining signal data relating to chemical reactions in a plurality of other wells within a region around the first well; and
performing steps (b) through (g) for each of the obtained signal data from the plurality of other wells to obtain multiple adjusted signal correction parameters.
3 . The method of claim 2 , further comprising calculating a region-wide estimate of the signal correction parameter using the multiple adjusted signal correction parameters.
4 . The method of claim 3 , wherein the comparing step comprises calculating a fitting metric that measures the fit between the predicted signals and the signal data from at least some of the plurality of wells.
5 . The method of claim 4 , wherein the fitting metric measures the fit between the predicted signals and the signal data from less than all of the plurality of wells; and
wherein the region-wide estimate excludes adjusted signal correction parameters from wells that produce a fitting metric exceeding a predetermined threshold.
6 . The method of claim 3 , further comprising performing a base calling analysis of the signal data from multiple wells within the region using the region-wide estimate of the signal correction parameter.
7 . The method of claim 1 , further comprising repeating steps (c) through (g) using the adjusted signal correction parameter.
8 . The method of claim 7 , wherein use of the adjusted signal correction parameter improves the fit between the signal data and the predicted signals.
9 . The method of claim 1 , wherein the phase-state model includes two or more signal correction parameters, including a carry forward rate and an incomplete extension rate.
10 . The method of claim 1 , wherein the comparing step comprises calculating a fitting metric that measures a fit between the predicted signals and the signal data.
11 . The method of claim 10 , wherein the adjusting step comprises determining a value of the signal correction parameter that optimizes the fitting metric.
12 . The method of claim 11 , wherein the fitting metric is calculated using only nucleotide flows that result in nucleotide non-incorporation or single nucleotide incorporations.
13 . The method of claim 1 , further comprising performing a base calling analysis of the signal data using the adjusted signal correction parameter.
14 . The method of claim 1 , wherein the set of nucleotide flows is a first set of nucleotide flows and the sequence information is a first sequence information, and further comprising:
applying the phase-state model using the adjusted signal correction parameter;
calculating, using the phase-state model, predicted signals resulting from a second set of nucleotide flows that includes nucleotide flows that are not in the first set of nucleotide flows;
making base calls by comparing the signal data to the predicted signals; and
obtaining a second sequence information about the polynucleotide strand, wherein the second sequence information includes sequence information not contained in the first sequence information.
15 . The method of claim 14 , further comprising repeating steps (d) through (g) using the second sequence information to obtain a further adjusted signal correction parameter.
16 . The method of claim 3 , wherein the region is a first region and wherein the phase-state model is adjusted for a signal droop rate that is obtained by a method comprising:
receiving signal data relating to chemical reactions in a plurality of wells within a second region of the array, wherein the plurality of wells includes the well containing the polynucleotide strand, wherein the second region is the same or different from the first region;
calculating a set of averaged signal values from the signal data; and
determining a region-wide signal droop rate by fitting a signal decay function to the set of averaged signal values.
17 - 19 . (canceled)
20 . A method of sequencing a polynucleotide strand contained in a well of an array having multiple wells, comprising:
receiving signal data relating to chemical reactions in a plurality of wells within a region of the array resulting from a flow of nucleotides to the array, wherein the plurality of wells includes the well containing the polynucleotide strand;
calculating a set of averaged signal values from the signal data, wherein the set of averaged signal values are stored in a machine-readable memory;
determining a region-wide signal droop rate by fitting a signal decay function to the set of averaged signal values; and
storing the region-wide signal droop rate in the memory.