Models for analyzing data from sequencing-by-synthesis operations

• US 10,146,906 B2
• Filed: 12/29/2011
• Issued: 12/04/2018
• Est. Priority Date: 12/30/2010
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Rearick, Todd, Schultz, Jonathan
• Primary Examiner(s): VANNI, GEORGE STEVEN
• Application Number:
  US13/339,846
• Publication Number:
  US 20120172241A1
• Time in Patent Office: 2,532 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/119 pH
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: G16B 30/20 Sequence assembly
• CPC Class Codes: G16B 40/00 ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
• CPC Class Codes: G16B 40/10 Signal processing, e.g. from mass spectrometry [MS] or from PCR

Abstract

Mathematical models for the analysis of signal data generated by sequencing of a polynucleotide strand using a pH-based method of detecting nucleotide incorporation(s). In an embodiment, the measured output signal from the reaction confinement region of a reactor array is mathematically modeled. The output signal may be modeled as a linear combination of one or more signal components, including a background signal component. This model is solved to determine the nucleotide incorporation signal. In another embodiment, the incorporation signal from the reaction confinement region of a reactor array is mathematically modeled.

Claims

1. A method of estimating a number of nucleotide incorporations when sequencing a polynucleotide strand, comprising:

flowing a series of nucleotide flows onto a reactor array comprising a plurality of chemFET sensors and having multiple reaction confinement regions, one or more copies of the polynucleotide strand being located in a loaded reaction confinement region of the reactor array, wherein the one or more copies of the polynucleotide strand are attached to a substrate particle;

receiving output signals from the plurality of chemFET sensors of the reactor array, the output signals being generated in response to chemical reactions between the one or more copies of the polynucleotide strand and the series of nucleotide flows;

determining a background signal for the loaded reaction confinement region using a model of an output signal from the loaded reaction confinement region, the model of the output signal from the loaded reaction confinement region being a linear combination of a function of a first signal component and a function of a second signal component, wherein determining the background signal comprises fitting the model to the received output signals based on at least a ratio parameter of the function of the second signal component, wherein;

the second signal component is generated based on an output signal from an unloaded reaction confinement region of the reactor array, and

the function of the second signal component comprises (i) a rate parameter that varies mathematically with a rate of change in an amount of hydrogen ions in the loaded reaction confinement region, and (ii) the ratio parameter, which ratio parameter varies mathematically with a ratio of a rate of change in the amount of hydrogen ions in the unloaded reaction confinement region relative to the rate of change in the amount of hydrogen ions in the loaded reaction confinement region;

determining an incorporation signal from the loaded reaction confinement region using the model of the output signal from the loaded reaction confinement region, wherein determining the incorporation signal further comprises fitting the model to at least some of the received output signals based on one or more parameters of the function of the first signal component; and

determining an estimate of a number of nucleotides incorporated into the polynucleotide strand using a method selection from (i) using a peak of the determined incorporation signal to estimate the number of nucleotides incorporated, and (ii) comparing the determined incorporation signal to a set of reference incorporation signal curves to estimate the number of nucleotides incorporated.

2. The method of claim 1, wherein the function of the second signal component comprises a product between the second signal component and the ratio parameter.

3. The method of claim 1, wherein the step of determining the background signal comprises fitting the model to background signal data from the loaded reaction confinement region using a method selected from the group comprising a regression analysis, a Bayesian technique, a least square analysis, and a Levenberg-Marquardt algorithm.

4. The method of claim 1, wherein at least some of the received output signals comprise signal data for a first nucleotide flow over the reactor array to the loaded reaction confinement region, wherein the first nucleotide flow results in a non-incorporation event in the loaded reaction confinement region.

5. The method of claim 4, wherein the first nucleotide flow over the reactor array is over a part of the polynucleotide strand located in the loaded reaction confinement region that is known to be non-complementary to a nucleotide of the first flow.

6. The method of claim 4, wherein a flow prior to the first nucleotide flow contains the same nucleotide as the first nucleotide flow.

7. The method of claim 4, wherein determining the incorporation signal comprises fitting the model to signal data for a second nucleotide flow to the loaded reaction confinement region to obtain the incorporation signal for the second nucleotide flow.

8. The method of claim 7, wherein the step of fitting the model to signal data for the second nucleotide flow comprises applying greater weight to a portion of the signal data that is earlier or later in time than another portion of the signal data.

9. The method of claim 4, wherein the function of the second signal component is derived from a flux of hydrogen ions between nucleotide reagent of the first nucleotide flow and the loaded reaction confinement region.

10. The method of claim 9, wherein the function of the second signal component is further derived from a total flux of hydrogen ions in the loaded reaction confinement region.

11. The method of claim 4, wherein the step of fitting the model to signal data for a first nucleotide flow comprises setting the first signal component to zero.

12. The method of claim 1, wherein the rate parameter is a product between a diffusion constant of the hydrogen ions in the loaded reaction confinement region and a buffering capacity of the loaded reaction confinement region.

13. The method of claim 12, wherein the ratio parameter that varies mathematically with the ratio of the rate of change in the amount of hydrogen ions in an unloaded reaction confinement region relative to the rate of change in the amount of hydrogen ions in the loaded reaction confinement region is a ratio of a product between a diffusion constant of the hydrogen ions in the unloaded reaction confinement region and a buffering capacity of the unloaded reaction confinement region relative to a product between the diffusion constant of the hydrogen ions in the loaded reaction confinement region and the buffering capacity of the loaded reaction confinement region.

14. The method of claim 13, wherein the fitting of the model for a first nucleotide flow comprises varying iteratively at least one of the rate parameter and the ratio parameter.

15. The method of claim 1, wherein the function of the second signal component further comprises a numerical approximation to an integral of a difference between the output signal of the unloaded reaction confinement region and the output signal from the loaded reaction confinement region over a period of time.

16. The method of claim 1, wherein the function of the first signal component comprises a buffering parameter relating to the buffering capacity of the loaded reaction confinement region.

17. The method of claim 16, wherein the fitting of the model to the signal data from the second nucleotide flow comprises varying iteratively the buffering parameter to fit with the signal data from the second nucleotide flow.

18. The method of claim 1, wherein determining an estimate of the number of nucleotides incorporated comprises using a peak of the determined incorporation signal to estimate the number of nucleotides incorporated.

19. The method of claim 1, wherein determining an estimate of the number of nucleotides incorporated comprises comparing the determined incorporation signal to a set of reference incorporation signal curves.

20. The method of claim 1, wherein determining the background signal comprises fitting the incorporation signal model to background signal data from the loaded reaction confinement region using an iterative process of varying the rate parameter and the ratio parameter to minimize a sum of residual errors between model-predicted signals and measured signals.

21. A non-transitory machine-readable storage medium comprising instructions which, when executed by a processor, cause the processor to perform a method of estimating a number of nucleotide incorporations when sequencing a polynucleotide strand comprising:

receiving output signals generated in response to chemical reactions resulting from a flow of a series of nucleotides onto a reactor array comprising a plurality of chemFET sensors and having multiple reaction confinement regions, one or more copies of the polynucleotide strand being located in a loaded reaction confinement region of the reactor array, wherein the one or more copies of the polynucleotide strand are attached to a substrate particle;

determining a background signal for the loaded reaction confinement region using a model of an output signal from the loaded reaction confinement region, the model of the output signal from the loaded reaction confinement region being a linear combination of a function of a first signal component and a function of a second signal component, wherein determining the background signal comprises fitting the model to the received output signals based on at least a ratio parameter of the function of the second signal component, wherein;

the second signal component is generated based on an output signal from an unloaded reaction confinement region of the reactor array, and

the function of the second signal comprises (i) a rate parameter that varies mathematically with a rate of change in the amount of hydrogen ions in the loaded reaction confinement region, and (ii) the ratio parameter, which ratio parameter varies mathematically with a ratio of a rate of change in the amount of hydrogen ions in the unloaded reaction confinement region relative to the rate of change in the amount of hydrogen ions in the loaded reaction confinement region;

determining an incorporation signal from the loaded reaction confinement region using the model of the output signal from the loaded reaction confinement region, wherein determining the incorporation signal further comprises fitting the model to at least some of the received output signals based on one or more parameters of the function of the first signal component;

determining an estimate of the number of nucleotides incorporated into the polynucleotide strand using a method selected from (i) using a peak of the determined incorporation signal to estimate the number of nucleotides incorporated, and (ii) comparing the determined incorporation signal to a set of reference incorporation signal curves to estimate the number of nucleotides incorporated; and

storing the estimate of the number of nucleotides incorporated in a data storage memory.

22. A method of estimating a number of nucleotide incorporations when sequencing a polynucleotide strand, comprising:

flowing a nucleotide reagent onto a reactor array comprising a plurality of chemFET sensors and having multiple reaction confinement regions, one or more copies of the polynucleotide strand being located in a loaded reaction confinement region of the reactor array;

receiving signal data from the plurality of chemFET sensors of the reactor array, the signal data being generated in response to chemical reactions between the one or more copies of the polynucleotide strand and nucleotides from the flowing of the nucleotide reagent;

processing the signal data from the loaded reaction confinement region by subtracting a background signal to obtain incorporation signal data;

fitting an incorporation signal model comprising a system of non-linear differential equations to the incorporation signal data obtained from the processing, the system of non-linear differential equations comprising an equation mathematically representing a rate of change in a nucleotide concentration in the loaded reaction confinement region and an equation mathematically representing an amount of active polymerase in the loaded reaction confinement region; and

determining, from the fit of the incorporation signal model, an estimate of a number of nucleotide incorporations resulting from the flowing of the nucleotide reagent.

23. The method of claim 22, wherein the incorporation signal model comprises an equation representing a flux of hydrogen ions generated in response to the chemical reactions between the one or more copies of the polynucleotide strand and nucleotides from the flowing of the nucleotide reagent.

24. The method of claim 22, wherein fitting the incorporation signal model to the incorporation signal data comprises applying greater weight to a portion of the signal data that is earlier or later in time than another portion of the signal data.

25. The method of claim 22, wherein the equation mathematically representing a rate of change in the nucleotide concentration in the loaded reaction confinement region comprises a difference between a first term that is proportional to a nucleotide concentration gradient and a second term that represents a rate at which nucleotides are consumed by the chemical reactions in the loaded reaction confinement region.

26. The method of claim 22, wherein the equation mathematically representing the amount of active polymerase in the loaded reaction confinement region comprises a sum of numbers of polymerases [a_n] located at polynucleotide strand positions where n additional base incorporations are needed to complete a homopolymer of length M, where n varies from 1 to M.

27. The method of claim 22, wherein the incorporation signal model further comprises an equation for the rate of change in the amount of active polymerase in the loaded reaction confinement region.

28. The method of claim 27, wherein the equation mathematically representing the rate of change in the amount of active polymerase in the loaded reaction confinement region comprises a term including a product between a nucleotide concentration and a number of polymerases [a_n] located at a polynucleotide strand position where n additional base incorporations are needed to complete a homopolymer of length M.

29. The method of claim 22, wherein the equation mathematically representing the amount of active polymerase in the loaded reaction confinement region comprises a cumulative Poisson equation that calculates a probability that any given polynucleotide strand has not yet completed all base incorporations in a homopolymer of length M.