Predictive model for use in sequencing-by-synthesis

• US 8,666,678 B2
• Filed: 10/27/2011
• Issued: 03/04/2014
• Est. Priority Date: 10/27/2010
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Davey, Melville, Meyer, Michael
• Primary Examiner(s): Brusca, John S
• Application Number:
  US13/283,320
• Publication Number:
  US 20120109598A1
• Time in Patent Office: 859 Days
• Field of Search: None
• US Class Current: 702/20
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 5/00 ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks

Abstract

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.

Claims

1. A sequencing method, comprising:

(a) flowing nucleotides onto a sensor array adapted for sequencing, the sensor array including a first well containing a polynucleotide strand;

(b) receiving from the sensor array signal data relating to chemical reactions resulting from the flow of nucleotides, the signal data comprising errors due to phase synchrony loss;

(c) determining, using the signal data, sequence information of at least a portion of the polynucleotide strand;

(d) 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;

(e) calculating, using the phase-state model, predicted signals resulting from the set of nucleotide flows;

(f) comparing the predicted signals to the signal data;

(g) adjusting the signal correction parameter of the phase-state model based on the comparison of the predicted signals to the signal data; and

(h) storing the adjusted signal correction parameter in the memory.

2. The method of claim 1, wherein the sensor array has multiple wells, 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 (c) through (h) 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 (d) through (h) 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 (e) through (h) 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 decay 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 first 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 decay rate by fitting a signal decay function to the set of averaged signal values.

17. A non-transitory machine-readable storage medium comprising instructions which, when executed by a processor, cause the processor to:

(a) receive signal data relating to chemical reactions resulting from a flow of nucleotides onto an array containing multiple wells, at least one of said wells containing a polynucleotide strand;

(b) determine, using the signal data, sequence information of at least a portion of the polynucleotide strand;

(c) construct 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 computer memory;

(d) calculate, using the phase-state model, predicted signals resulting from the set of nucleotide flows;

(e) compare the predicted signals to the signal data;

(f) adjust the signal correction parameter of the phase-state model based on the comparison of the predicted signals to the signal data; and

(g) store the adjusted signal correction parameter in the memory.

18. An apparatus comprising:

a machine-readable memory; and

a processor configured to execute machine-readable instructions, said instructions which when executed cause the apparatus to;

(a) receive signal data relating to chemical reactions resulting from a flow of nucleotides onto an array containing multiple wells, at least one of said wells containing a polynucleotide strand;

(b) determine, using the signal data, sequence information of at least a portion of the polynucleotide strand;

(c) construct 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 the memory;

(d) calculate, using the phase-state model, predicted signals resulting from the set of flows;

(e) compare the predicted signals to the signal data;

(f) adjust the signal correction parameter of the phase-state model based on the comparison of the predicted signals to the signal data; and

(g) store the adjusted signal correction parameter in the memory.

19. The apparatus of claim 18, further comprising:

a plurality of reservoirs comprising different nucleotide reagents; and

a flow chamber for receiving the nucleotide reagents.

20. A sequencing method, comprising:

flowing nucleotides onto an array adapted for sequencing, the array having multiple wells, the wells comprising sensors configured to measure physical or chemical parameters providing information about a chemical reaction taking place in the wells, and the wells including a well containing a polynucleotide strand;

receiving from the array signal data relating to chemical reactions in a plurality of wells within a region of the array resulting from the flow of nucleotides to the array, wherein the plurality of wells within the region of the array 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 decay rate by fitting a signal decay function to the set of averaged signal values; and

storing the region-wide signal decay rate in the memory.