Method and program for performing baseline correction of amplification curves in a PCR experiment

• US 8,473,216 B2
• Filed: 11/30/2007
• Issued: 06/25/2013
• Est. Priority Date: 11/30/2006
• Status: Active Grant

• Current Assignee: Fluidigm Corporation
• Original Assignee: Fluidigm Corporation
• Inventors: Sun, Gang, Ramakrishnan, Ramesh, Jones, Robert C.
• Primary Examiner(s): Whaley, Pablo S
• Application Number:
  US11/948,782
• Publication Number:
  US 20080129736A1
• Time in Patent Office: 2,034 Days
• Field of Search: None
• US Class Current: 702/19
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

A method of adjusting amplification curves in a PCR experiment includes receiving a plurality of amplification curves for a sample and computing a first parameter for each of the plurality of amplification curves. The method also includes computing a second parameter for each of the plurality of amplification curves and computing a third parameter using at least a portion of the first or second parameters. The method further includes computing an offset for each of the plurality of amplification curves. The offset is a function of the first parameter and the third parameter. Moreover, the method includes adjusting at least one of the plurality of amplification curves by subtracting the offset.

Claims

1. A method of performing baseline correction for amplification curves in a PCR experiment, the method comprising:

providing a processor;

receiving a plurality of amplification curves for a sample, wherein the plurality of amplification curves are characterized by a baseline drift, each of the plurality of amplification curves being associated with a sample and one of a plurality of reagents;

computing, using the processor, a first parameter for each of the plurality of amplification curves;

computing, using the processor, a slope value for each of the plurality of amplification curves;

computing, using the processor, a third parameter equal to an average of the slope values associated with each of the plurality of amplification curves;

computing, using the processor, a baseline correction value for each of the plurality of amplification curves, the baseline correction value being a function of the first parameter and the third parameter; and

performing baseline correction, using the processor, for at least one of the plurality of amplification curves by subtracting the baseline correction value.

2. The method of claim 1 wherein computing a first parameter for each of the plurality of amplification curves comprises averaging K amplification values for each curve.

3. The method of claim 1 wherein computing a slope value comprises using a linear regression analysis.

4. The method of claim 3 wherein computing a slope value further comprises using amplification values that fall below a predetermined value.

5. The method of claim 4 wherein the predetermined value is associated with a linear portion of each of the plurality of amplification curves.

6. The method of claim 4 wherein the predetermined value is associated with a Ct estimate line.

7. The method of claim 4 wherein computing a slope value further comprises using amplification values from a substantially linear portion of the curve.

8. The method of claim 1 wherein computing a third parameter comprises averaging a portion of the slope values.

9. The method of claim 8 wherein the portion of the slope values exhibit a goodness of fit under a linear regression analysis that is greater than a predetermined threshold.

10. The method of claim 9 wherein the predetermined threshold is greater than or equal to 0.98.

11. The method of claim 1 wherein the slope value is computed using a predetermined number of data values.

12. The method of claim 11 wherein the predetermined number of data values is greater than or equal to four.

13. The method of claim 1 wherein the baseline correction value is a function equal to the first parameter added to the third parameter times an index.

14. The method of claim 13 wherein the index is equal to a cycle number minus one.

15. The method of claim 1 wherein the sample is a pre-amplified DNA sample.

16. The method of claim 1 wherein each of the plurality of amplification curves represents a ratio of a reporter dye signal to a passive reference dye signal as a function of PCR cycle number.

17. The method of claim 1 wherein the PCR experiment is performed using a microfluidic device comprising an array of reaction chambers larger than or equal to 48×

48 chambers.

18. The method of claim 17 wherein the array of reaction chambers comprises a plurality of reaction chambers characterized by a volume of less than or equal to 10 nanoliters.

19. The method of claim 1 further comprising:

receiving an initial set of amplification curves for the sample; and

selecting a subset of the initial set to provide the plurality of amplification curves for the sample.

20. A non-transitory computer-readable storage medium including a plurality of computer-readable instructions tangibly embodied on the computer-readable storage medium, which, when executed by a data processor, provide for adjusting amplification curves in a PCR experiment, the computer-readable storage medium including:

instructions that cause the data processor to receive a plurality of amplification curves for a sample, each of the plurality of amplification curves being associated with a sample and one of a plurality of reagents;

instructions that cause the data processor to compute a first parameter for each of the plurality of amplification curves;

instructions that cause the data processor to compute a slope value for each of the plurality of amplification curves;

instructions that cause the data processor to compute a third parameter equal to an average of the slope values associated with each of the plurality of amplification curves;

instructions that cause the data processor to compute a baseline correction value for each of the plurality of amplification curves, the baseline correction value being a function of the first parameter and the third parameter; and

instructions that cause the data processor to perform baseline correction for at least one of the plurality of amplification curves by subtracting the baseline correction value.

28. The non-transitory computer-readable storage medium of claim 20 wherein the first parameter for each of the plurality of amplification curves comprises an average of K amplification values for each curve.

29. The non-transitory computer-readable storage medium of claim 20 wherein the baseline correction value is a function equal to the first parameter added to the third parameter times an index.

30. The non-transitory computer-readable storage medium of claim 29 wherein the index is equal to a cycle number minus one.

31. The non-transitory computer-readable storage medium of claim 20 further comprising instructions that cause the data processor to perform the PCR experiment using a microfluidic device comprising an array of reaction chambers larger than or equal to 48×

48 chambers.

32. The non-transitory computer-readable storage medium of claim 31 wherein the array of reaction chambers comprises a plurality of reaction chambers characterized by a volume of less than or equal to 10 nanoliters.

21. A method for performing baseline correction for ratio curves in a PCR experiment, the method comprising:

providing a processor;

obtaining a first series of amplification values for a DNA sample, wherein the DNA sample is associated with a sample and a first of a plurality of reagents;

averaging, using the processor, J values of the first series of amplification values to provide a first baseline value;

computing, using the processor, a first slope using at least a portion of the first series of amplification values;

obtaining a second set of amplification values for the DNA sample, wherein the DNA sample is associated with a sample and a second of a plurality of reagents;

averaging, using the processor, K values of the second series of amplification values to provide a second baseline value;

computing, using the processor, a second slope using at least a portion of the second series of amplification values;

averaging, using the processor, the first slope and the second slope to provide an average slope for the DNA sample;

computing, using the processor, a first baseline correction function equal to the first baseline value plus the average slope times an index;

computing, using the processor, a second baseline correction function equal to the second baseline value plus the average slope times the index;

performing baseline correction, using the processor, the first series of amplification values by subtracting the first baseline correction function from the first series of amplification values; and

performing baseline correction, using the processor, the second series of amplification values by subtracting the second baseline correction function from the second series of amplification values.

22. The method of claim 21 wherein computing the first slope comprises using a linear regression analysis.

23. The method of claim 22 wherein computing the first slope further comprises using amplification values that fall below a predetermined value associated with a linear portion of the first series of amplification values for the DNA sample.

24. The method of claim 21 wherein the index is equal to a cycle number minus one.

25. The method of claim 21 wherein the DNA sample is a pre-amplified DNA sample.

26. The method of claim 21 wherein the PCR experiment is performed using a microfluidic device comprising an array of reaction chambers larger than or equal to 48×

48 chambers.

27. The method of claim 26 wherein the array of reaction chambers comprises a plurality of reaction chambers characterized by a volume of less than or equal to 10 nanoliters.