Time-warped background signal for sequencing-by-synthesis operations

• US 9,594,870 B2
• Filed: 12/29/2011
• Issued: 03/14/2017
• Est. Priority Date: 12/29/2010
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Hubbell, Earl
• Primary Examiner(s): Riggs, II, Larry D
• Application Number:
  US13/339,753
• Publication Number:
  US 20120173158A1
• Time in Patent Office: 1,902 Days
• US Class Current: 1/1
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• 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

Abstract

Methods for analyzing signal data generated by sequencing of a polynucleotide strand using a pH-based method of detecting nucleotide incorporation(s). In an embodiment, the method comprises formulating a function that models the output signal of a representative empty well of a reactor array. A time transformation is applied to the empty well function to obtain a time-warped empty well function. The time-warped empty well function is fitted to an output signal from the loaded well representative of a flow that results in a non-incorporation event in the loaded well. The fitted time-warped empty well function can then be used to analyze output signals from the loaded well for other flows.

Claims

1. A method of sequencing a polynucleotide strand using a sequencing apparatus and reactor array adapted to perform sequencing-by-synthesis operations, comprising:

flowing a series of nucleotide reagents onto a reactor array from an inlet to an outlet, wherein the reactor array has multiple reaction confinement regions having different signal responses due to their different locations along a flow path of the nucleotide reagents from the inlet to the outlet, wherein the polynucleotide strand is located in a loaded reaction confinement region of the reactor array;

receiving signal data relating to chemical reactions resulting from the flowing of the series of nucleotide reagents onto the reactor array having multiple reaction confinement regions, wherein the signal data include an output signal of an empty reaction confinement region and an output signal of the loaded reaction confinement region;

determining an empty reaction confinement region function that models the output signal of a representative empty reaction confinement region;

determining a time-warped empty reaction confinement region function by applying a time transformation to the empty reaction confinement region function, wherein the time transformation changes a time scale of the empty reaction confinement region function thereby adjusting for systematic time lag differences due to the location of the representative empty reaction confinement region along the flow path of the nucleotide reagents from the inlet to the outlet;

applying the time-warped empty reaction confinement region function to the output signal of the loaded reaction confinement region detected during the flowing to generate a corrected output signal; and

analyzing the corrected output signal to estimate a number of nucleotide incorporations having occurred for the polynucleotide strand located in the loaded reaction confinement region in response to the flowed nucleotide reagents.

2. The method of claim 1, further comprising fitting the time-warped empty reaction confinement region function to an output signal from the loaded reaction confinement region that is representative of a first flow, wherein the first flow results in a non-incorporation event in the loaded reaction confinement region, to produce a fitted time-warped empty reaction confinement region function.

3. The method of claim 2, further comprising applying the fitted time-warped empty reaction confinement region function to an output signal for a second flow to the loaded reaction confinement region to obtain a corrected output signal for the second flow, wherein the corrected output signal for the second flow is analyzed to estimate the number of nucleotides incorporated into the polynucleotide strand.

4. The method of claim 1, wherein the time transformation is a polynomial function of time.

5. The method of claim 4, wherein the time transformation is a quadratic or cubic polynomial function of time.

6. The method of claim 1, wherein the time transformation is a linear function of time.

7. The method of claim 1, wherein the step of determining the empty reaction confinement region function comprises performing a spline fitting of the output signal from the representative empty reaction confinement region.

8. The method of claim 1, wherein the empty reaction confinement region function is a polynomial function of time.

9. The method of claim 1, wherein the empty reaction confinement region function is an exponential function of time.

10. The method of claim 1, wherein the representative empty reaction confinement region represents a plurality of empty reaction confinement regions within a region that includes the loaded reaction confinement region.

11. The method of claim 1, wherein the reactor array includes a chemFET sensor array for detecting hydrogen ions in the reaction confinement regions of the array.

12. The method of claim 3, further comprising fitting the time-warped empty reaction confinement region function to an output signal from the loaded reaction confinement region representative of a third flow, wherein the third flow occurs later than the second flow and results in a non-incorporation event in the loaded reaction confinement region.

13. The method of claim 3, wherein the time transformation comprises a parameter, and further comprising:

obtaining an output signal from the loaded reaction confinement region that is representative of a third flow, wherein the third flow occurs later than the second flow and results in a non-incorporation event in the loaded reaction confinement region;

obtaining a derivative of the output signal for the third flow; and

adjusting the parameter of the time transformation using the derivative.

14. The method of claim 3, wherein the polynucleotide strand includes a portion having a known sequence, and wherein the first flow is over the portion having the known sequence.

15. The method of claim 3, wherein the step of fitting the time-warped empty reaction confinement region function comprises minimizing a difference between the output signal from the loaded reaction confinement region and a signal predicted by the time-warped empty reaction confinement region function.

16. The method of claim 3, wherein the corrected output signal for the second flow is obtained by subtracting the fitted time-warped empty reaction confinement region function from the output signal for the second flow.

17. The method of claim 3, wherein the corrected output signal is obtained by solving a model for an output signal model for from the loaded reaction confinement region, wherein the output signal model comprises a background component and an incorporation signal component, wherein the background component is the fitted time-warped empty reaction confinement region function.

18. The method of claim 3, wherein the analyzing the corrected output signal further comprises:

comparing the corrected output signal to a library of incorporation signal shapes comprising multiple signal shapes that are associated with different n-mer lengths; and

based on the comparison, determining an estimate of the number of nucleotides incorporated into the polynucleotide strand.

19. The method of claim 18, further comprising using a catenary multi-compartment model to add to or generate or allow cross-referencing to the library of incorporation signal shapes, wherein the multi-compartment model comprises a series of two or more compartments that represent molecular locations on the homopolymer length.

20. The method of claim 3, further comprising:

determining a function for an incorporation signal, wherein the function includes a parameter for the n-mer length;

fitting the incorporation signal function to the corrected output signal to solve for the parameter for the n-mer length; and

using the parameter for the n-mer length to estimate the number of nucleotides incorporated into the polynucleotide strand.

21. The method of claim 20, further comprising using a catenary multi-compartment model to determine the function for the incorporation signal.

22. The method of claim 1, further comprising using the time-warped empty reaction confinement region function to determine whether the loaded reaction confinement region is an outlier reaction confinement region.

25. The method of claim 1, further comprising applying the time-warped empty reaction confinement region function to a subsequent output signal of the loaded reaction confinement region in response to a subsequent flow to produce a corrected output signal for the subsequent flow.

23. A sequencing apparatus for sequencing a polynucleotide strand using sequencing-by-synthesis, comprising:

a reactor array having multiple reaction confinement regions;

a fluidic interface configured to flow nucleotide reagents onto the reactor array from an inlet to an outlet along a flow path, wherein the multiple reaction confinement regions have different signal responses due to their different locations along the flow path;

a machine-readable memory; and

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

flow a series of nucleotide reagents onto the reactor array from the inlet to the outlet, wherein the polynucleotide strand is located in a loaded reaction confinement region of the reactor array, resulting in one or more nucleotide incorporations in the polynucleotide strand;

receive signal data relating to chemical reactions resulting from the flow of the series of nucleotide reagents onto the reactor array having multiple reaction confinement regions, wherein the signal data include an output signal of an empty reaction confinement region and an output signal of the loaded reaction confinement region;

determine an empty reaction confinement region function that models the output signal of a representative empty reaction confinement region, wherein the empty reaction confinement region function is stored in a computer memory;

determine a time-warped empty reaction confinement region function by applying a time transformation to the empty reaction confinement region function, wherein the time transformation changes a time scale of the empty reaction confinement region function, thereby adjusting for systematic time lag differences due to the location of the representative empty reaction confinement region along the flow path of the nucleotide reagents from the inlet to the outlet;

apply the time-warped empty reaction confinement region function to the output signal of the loaded reaction confinement region detected during the flow to generate a corrected output signal;

analyze the corrected output signal to estimate a number of nucleotide incorporations having occurred for the polynucleotide strand located in the loaded reaction confinement region in response to flowed nucleotide reagents; and

store the estimated number of nucleotide incorporations in the computer memory.

24. A non-transitory machine-readable storage medium comprising instructions which, when executed by a processor with a sequencing apparatus and reactor array adapted to perform sequencing-by-synthesis operations, cause the processor and sequencing apparatus to sequence a polynucleotide strand using sequencing-by-synthesis by performing the following steps:

flow a series of nucleotide reagents onto the reactor array from an inlet to an outlet, wherein the reactor array has multiple reaction confinement regions having different signal responses due to their different locations along a flow path of the nucleotide reagents from the inlet to the outlet, wherein the polynucleotide strand is located in a loaded reaction confinement region of the reactor array;

receive signal data relating to chemical reactions resulting from the flow of the series of nucleotide reagents onto the reactor array having multiple reaction confinement regions, wherein the signal data include an output signal of an empty reaction confinement region and an output signal of the loaded reaction confinement region;

determine an empty reaction confinement region function that models the output signal of a representative empty reaction confinement region, wherein the empty reaction confinement region function is stored in a computer memory;

determine a time-warped empty reaction confinement region function by applying a time transformation to the empty reaction confinement region function, wherein the time transformation changes a time scale of the empty reaction confinement region function, thereby adjusting for systematic time lag differences due to the location of the representative empty reaction confinement region along the flow path of the nucleotide reagents from the inlet to the outlet;

apply the time-warped empty reaction confinement region function to the output signal of the loaded reaction confinement region detected during the flow to generate a corrected output signal;

analyze the corrected output signal to estimate a number of nucleotide incorporations having occurred for the polynucleotide strand located in the loaded reaction confinement region in response to flowed nucleotide reagents; and

store the estimated number of nucleotide incorporations in the computer memory.