MODELS FOR ANALYZING DATA FROM SEQUENCING-BY-SYNTHESIS OPERATIONS

• US 20120172241A1
• Filed: 12/29/2011
• Published: 07/05/2012
• 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
• Granted Patent: US 10,146,906 B2
• Time in Patent Office: Days
• US Class Current: 506/7
• 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 sequencing a polynucleotide strand, comprising:

flowing a series of nucleotide flows onto a reactor array 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 output signals from the reactor array;

determining a background signal for the loaded reaction confinement region using a model of an output signal from the loaded reaction confinement region;

determining an incorporation signal from the loaded reaction confinement region using the model of an output signal from the loaded reaction confinement region; and

determining an estimate of a number of nucleotides incorporated into the polynucleotide strand.

2. The method of claim 1, wherein the model comprises a function for a background signal component and a function for an incorporation signal component.

3. The method of claim 2, wherein the step of determining the background signal comprises fitting the model to background signal data from the loaded reaction confinement region.

4. The method of claim 3, wherein the background signal data comprises representative signal data for a first flow to the loaded reaction confinement region, wherein the first flow results in a non-incorporation event in the loaded reaction confinement region.

5. The method of claim 4, wherein the first flow is over a part of the polynucleotide strand that is known to be non-complementary to the nucleotide in the first flow.

6. The method of claim 4, wherein the first flow is immediately preceded by a prior flow that contains the same nucleotide as in the first flow.

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

8. The method of claim 7, wherein the step of fitting the model to signal data for the second 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 7, wherein the function for the background signal component comprises a rate parameter relating to a rate of change in the amount of hydrogen ions in the loaded reaction confinement region.

10. The method of claim 9, wherein the function for the background signal component further comprises a ratio parameter relating to a ratio of a rate of change in the amount of hydrogen ions in a representative empty reaction confinement region relative to the rate of change in the amount of hydrogen ions in the loaded reaction confinement region.

11. The method of claim 7, wherein the function for the background signal component comprises the difference between the output signal of a representative empty reaction confinement region and the output signal from the loaded reaction confinement region.

12. The method of claim 11, wherein the function for the background signal component comprises the integral of the difference between the output signal of a representative empty reaction confinement region and the output signal from the loaded reaction confinement region from one or more prior time frames.

13. The method of claim 7, wherein the function for the background signal component is derived from the flux of hydrogen ions between the nucleotide reagent of the first flow and the loaded reaction confinement region.

14. The method of claim 13, wherein the function for the background signal component is further derived from the total flux of hydrogen ions in the loaded reaction confinement region.

15. The method of claim 14, wherein the function for the background signal component is further derived from the flux of hydrogen ions between the nucleotide reagent of the first flow and the representative empty reaction confinement region.

16. The method of claim 10, wherein the fitting of the model for the first flow comprises varying at least one of the rate parameter and the ratio parameter to improve the fit to the signal data from the first flow.

17. The method of claim 7, wherein the function for the incorporation signal component comprises a buffering parameter relating to the buffering capacity of the loaded reaction confinement region.

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

19. The method of claim 7, wherein the step of fitting the model to representative signal data for the first flow comprises setting the incorporation signal component to zero or near zero.

20. The method of claim 1, wherein the step of determining an estimate of the number of nucleotides incorporated further comprises fitting the incorporation signal model to the incorporation signal.

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

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

receive output signals relating to chemical reactions resulting from the flow of a series of nucleotides onto a reactor array 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;

determine a background signal for the loaded reaction confinement region using a model of an output signal from the loaded reaction confinement region, wherein the model is stored in a computer memory;

determine an incorporation signal from the loaded reaction confinement region using the model of an output signal from the loaded reaction confinement region;

determine an estimate of the number of nucleotides incorporated into the polynucleotide strand; and

store the estimate of the number of nucleotides incorporated in the memory.

23. A method of sequencing a polynucleotide strand, comprising:

flowing a nucleotide reagent onto a reactor array 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 reactor array;

processing the signal data from the loaded reaction confinement region to obtain incorporation signal data; and

determining an estimate of a number of nucleotide incorporations resulting from the nucleotide reagent flow by applying an incorporation signal model to the incorporation signal data.

24. The method of claim 23, wherein the incorporation signal model comprises an equation for the flux of hydrogen ions generated by a nucleotide incorporation reaction.

25. The method of claim 24, wherein the step of determining an estimate of the number of nucleotide incorporations comprises fitting the incorporation signal model to the incorporation signal data.

26. The method of claim 25, wherein the step of 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.

27. The method of claim 25, wherein the step of determining an estimate of the number of nucleotide incorporations further comprises using the results of the fitting to estimate the number of nucleotide incorporations.

28. The method of claim 27, wherein the model further comprises an equation for a rate of change in the nucleotide amount in the loaded reaction confinement region.

29. The method of claim 27, wherein the model further comprises an equation for the amount of active polymerase in the reaction confinement region.

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