System and method for correcting primer extension errors in nucleic acid sequence data

• US 8,301,394 B2
• Filed: 02/15/2007
• Issued: 10/30/2012
• Est. Priority Date: 02/16/2006
• Status: Active Grant

• Current Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Original Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Inventors: Chen, Yi-Ju, McDade, Keith, Simpson, John
• Primary Examiner(s): Brusca, John S
• Application Number:
  US12/224,065
• Publication Number:
  US 20100192032A1
• Time in Patent Office: 2,084 Days
• Field of Search: 714/758, 435/6.1, 435/6.19, 702/19, 702/20, 702/22, 702/27
• US Class Current: 702/20
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• 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 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

An embodiment of method for correcting an error associated with phasic synchrony of sequence data generated from a population of substantially identical copies of a template molecule is described that comprises (a) detecting a signal generated in response to an incorporation of one or more nucleotides in a sequencing reaction; (b) generating a value for the signal; and (c) correcting the value for the phasic synchrony error using a first parameter and a second carry forward parameter.

Claims

1. A method for correcting an error associated with phasic synchrony of sequence data generated from a population of substantially identical copies of a template nucleic acid molecule, comprising:

(a) detecting a signal generated in response to an incorporation of one or more nucleotides in a sequencing reaction, wherein the signal is generated from the population of substantially identical copies of the template nucleic acid molecule;

(b) generating a signal value for the signal;

(c) repeating steps (a)-(b) for a plurality of sequence positions of the template nucleic acid molecule;

(d) correcting a plurality of the signal values for a phasic synchrony error using a first parameter representative of an incomplete extension component of the phasic synchrony error that comprises a measure of one or more of the substantially identical copies of the template nucleic acid molecules behind an extension phase of the population and a second parameter representative of a carry forward component of the phasic synchrony error that comprises a measure of one or more of the substantially identical copies of the template nucleic acid molecules ahead of the extension phase of the population.

2. The method of claim 1, further comprising:

(e) incorporating a plurality of the signal values into a representation of the template molecule.

3. The method of claim 2, wherein:

the representation includes a flowgram.

4. The method of claim 1, wherein:

the incomplete extension component and the carry forward component that are both treated as substantially constant for the plurality of sequence positions of the template molecule.

5. The method of claim 1, wherein:

the carry forward component is treated as substantially constant for the plurality of sequence positions of the template molecule.

6. The method of claim 1, wherein:

the signal includes light emitted in response to the incorporation of the one or more nucleotides.

7. The method of claim 6, wherein:

the light includes chemiluminescent light from the sequencing reaction.

8. The method of claim 7, wherein:

the sequencing reaction includes a pyrophosphate sequencing reaction.

9. The method of claim 6, wherein:

the light includes fluorescent light from the sequencing reaction.

10. The method of claim 9, wherein:

the sequencing reaction includes a sequencing reaction that employs reversible terminators.

11. The method of claim 1, wherein:

the signal value is indicative of a number of the incorporated nucleotides.

12. The method of claim 1, wherein:

the first parameter and the second parameter are estimated by searching for a best fit to a matrix equation.

13. The method of claim 12, wherein:

the estimation of the best fit for the first and second parameters values include searching using an interval between test values and applying one or more approximations to the matrix equation at each test value, wherein the approximations provide for improved computational efficiency of the estimation.

38. The method of claim 1, wherein:

the step of correcting comprises inverting the signal values to give a theoretical signal value using a matrix equation.

14. A method for correcting an error associated with phasic synchrony of sequence data generated from a population of substantially identical copies of a template nucleic acid molecule, comprising:

(a) detecting a signal generated in response to an incorporation of one or more nucleotides in a sequencing reaction, wherein the signal is generated from the population of substantially identical copies of the template nucleic acid molecule;

(b) generating a signal value for the signal;

(c) incorporating the signal value into a representation associated with a sequence of a template nucleic acid molecule;

(d) repeating steps (a)-(c) for a plurality of sequence positions of the template nucleic acid molecule;

(e) correcting a plurality of the signal values for phasic synchrony error in the representation using a first parameter representative of an incomplete extension component of the phasic synchrony that comprises a measure of one or more of the substantially identical copies of the template nucleic acid molecules behind an extension phase of the population and a second parameter representative of a carry forward component of the phasic synchrony error that comprises a measure of one or more of the substantially identical copies of the template nucleic acid molecules ahead of the extension phase of the population; and

(f) generating a corrected representation using the corrected signal values.

15. The method of claim 14, further comprising:

(g) iteratively repeating steps (e)-(f) using the corrected signal values from a previous iteration for step (e), wherein some or all of the corrected signal values improve in quality with each iteration.

16. The method of claim 14, wherein:

the incomplete extension component and the carry forward component that are both treated as substantially constant for the plurality of sequence positions of the template molecule.

17. The method of claim 14, wherein:

the carry forward component is treated as substantially constant for the plurality of sequence positions of the template molecule.

18. The method of claim 14, wherein:

the signal includes light emitted in response to the incorporation of the one or more nucleotides.

19. The method of claim 18, wherein:

the light includes chemiluminescent light from the sequencing reaction.

20. The method of claim 19, wherein:

the sequencing reaction includes a pyrophosphate sequencing reaction.

21. The method of claim 18, wherein:

the light includes fluorescent light from the sequencing reaction.

22. The method of claim 21, wherein:

the sequencing reaction includes a sequencing reaction that employs reversible terminators.

23. The method of claim 14, wherein:

the signal value is indicative of a number of the incorporated nucleotides.

24. The method of claim 14, wherein:

a value for the first parameter and a value for the second parameter are estimated by searching for a best fit to a matrix equation for each of the first and second parameters.

25. The method of claim 24, wherein:

the estimation of the best fit for the first and second parameters include searching using an interval between test values and applying one or more approximations on a matrix equation at each test value, wherein the approximations provide for improved computational efficiency of the estimation.

26. The method of claim 14, wherein:

the representation and corrected representation include a flowgram.

27. A method for correcting an error associated with phasic synchrony of sequence data generated from a population of substantially identical copies of a template nucleic acid molecule, comprising:

(a) detecting a signal generated in response to an incorporation of one or more nucleotides in a sequencing reaction, wherein the signal is generated from the population of substantially identical copies of the template nucleic acid molecules;

(b) generating a signal value for the signal;

(c) incorporating the signal value into a representation associated with a sequence of the template nucleic acid molecule;

(d) repeating steps (a)-(c) for a plurality of sequence positions of the template molecule;

(e) dividing the representation into a plurality of sub-sets, wherein each sub-set comprises one or more of the sequence positions of the template molecule;

(f) estimating a value for a first parameter representative of an incomplete extension component of a phasic synchrony error that comprises a measure of one or more of the substantially identical copies of the template nucleic acid molecules behind an extension phase of the population and a value for a second parameter representative of a carry forward component of the phasic synchrony error that comprises a measure of one or more of the substantially identical copies of the template nucleic acid molecules ahead of the extension phase of the population in each sub-set;

(g) correcting the signal values in each sub-set for the phasic synchrony error using the estimations for the first parameter value and for the second parameter value for each respective sub-set; and

(h) combining the corrected sub-sets into a corrected representation using the corrected signal values.

28. The method of claim 27, wherein:

the incomplete extension component and the carry forward component fluctuate over a plurality of the sequence positions of the template nucleic acid molecule.

29. The method of claim 27, wherein:

the carry forward component fluctuates over a plurality of the sequence positions of the template nucleic acid molecule.

30. A system for correcting an error associated with phasic synchrony of sequence data generated from a population of substantially identical copies of a template nucleic acid molecule, comprising:

a computer comprising program code stored for execution thereon, the program code performing a method comprising;

(a) generating a value for a signal detected in response to an incorporation of one or more nucleotides in a sequencing reaction for a plurality of sequence positions of the template nucleic acid molecule, wherein the signal is generated from the population of substantially identical copies of the template nucleic acid molecules; and

(b) correcting a plurality of the values for the plurality of sequence positions of the template nucleic acid molecule for a phasic synchrony error using a first parameter representative of an incomplete extension component of the phasic synchrony error that comprises a measure of one or more of the substantially identical copies of the template nucleic acid molecules behind an extension phase of the population and a second parameter representative of a carry forward component of the phasic synchrony error that comprises a measure of one or more of the substantially identical copies of the template nucleic acid molecules ahead of the extension phase of the population.

31. The system of claim 30, wherein the method performed by the program code further comprises:

(d) incorporating a plurality of the values into a representation of the template molecule.

32. The system of claim 31, wherein the method performed by the program code further comprises:

(e) providing the representation to a user.

33. A system for correcting an error associated with phasic synchrony of sequence data generated from a population of substantially identical copies of a template nucleic acid molecule, comprising:

a computer comprising program code stored for execution thereon, the program code performing a method comprising;

(a) generating a value for a signal detected in response to an incorporation of one or more nucleotides in a sequencing reaction for a plurality of sequence positions of the template nucleic acid molecule, wherein the signal is generated from the population of substantially identical copies of the template nucleic acid molecules;

(b) incorporating the value into a representation associated with a sequence of a template nucleic acid molecule;

(c) repeating steps (a)-(b) for a plurality of sequence positions of the template nucleic acid molecule;

(d) correcting a plurality of the values for phasic synchrony error in the representation using a first parameter representative of an incomplete extension component of the phasic synchrony error that comprises a measure of one or more of the substantially identical copies of the template nucleic acid molecules behind an extension phase of the population and a second parameter representative of a carry forward component of the phasic synchrony error that comprises a measure of one or more of the substantially identical copies of the template nucleic acid molecules ahead of the extension phase of the population; and

(e) generating a corrected representation using the corrected values.

34. The system of claim 33, wherein the method performed by the program code further comprises:

(f) iteratively repeating steps (d)-(e) using the corrected values from a previous iteration for step (d), wherein some or all of the corrected values improve in quality with each iteration.

35. The system of claim 34, wherein:

the step of iteratively repeating is responsive to a user selection of a number of iterations to perform.

36. The system of claim 33, wherein the method performed by the program code further comprises:

(f) providing the corrected representation to a user.

37. A system for correcting an error associated with phasic synchrony of sequence data generated from a population of substantially identical copies of a template nucleic acid molecule, comprising:

a computer comprising program code stored for execution thereon, the program code performing a method comprising;

(a) generating a value for a signal detected in response to an incorporation of one or more nucleotides in a sequencing reaction for a plurality of sequence positions of the template nucleic acid molecule, wherein the signal is generated from the population of substantially identical copies of the template nucleic acid molecules;

(b) incorporating the value into a representation associated with a sequence of the template nucleic acid molecule;

(c) repeating steps (a)-(c) for a plurality of sequence positions of the template molecule;

(d) dividing the representation into a plurality of sub-sets, wherein each sub-set comprises one or more of the sequence positions of the template molecule;

(e) estimating a value for a first parameter representative of an incomplete extension component of a phasic synchrony error that comprises a measure of one or more of the substantially identical copies of the template nucleic acid molecules behind an extension phase of the population and a value for a second parameter representative of a carry forward component of the phasic synchrony error that comprises a measure of one or more of the substantially identical copies of the template nucleic acid molecules ahead of the extension phase of the population in each sub-set;

(f) correcting the signal values in each sub-set for the phasic synchrony error using the estimations for the first parameter value and for the second parameter value for each respective sub-set; and

(g) combining the corrected sub-sets into a corrected representation using the corrected values.