METHOD AND APPARATUS FOR BIOLOGICAL SAMPLE ANALYSIS

• US 20080129736A1
• Filed: 11/30/2007
• Published: 06/05/2008
• Est. Priority Date: 11/30/2006
• Status: Active Grant

• Current Assignee: Fluidigm Corporation
• Original Assignee: Fluidigm Corporation
• Inventors: Sun, Gang, Jones, Robert C., Ramakrishnan, Ramesh
• Granted Patent: US 8,473,216 B2
• Time in Patent Office: Days
• US Class Current: 345/442
• 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 adjusting amplification curves in a PCR experiment, the method comprising:

receiving a plurality of amplification curves for a sample;

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

computing a second parameter for each of the plurality of amplification curves;

computing a third parameter using at least a portion of the first or second parameters;

computing an offset for each of the plurality of amplification curves, the offset being a function of the first parameter and the third parameter; and

adjusting at least one of the plurality of amplification curves by subtracting the offset.

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 second parameter for each of the plurality of amplification curves comprises computing a slope value for each curve.

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

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

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

7. The method of claim 5 wherein the predetermined value is associated with a Ct estimate line.

8. The method of claim 5 wherein computing a slope value further comprises using amplification values from a substantially linear portion of the curve.

9. The method of claim 3 wherein computing a third parameter comprises averaging a portion of the slope values.

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

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

12. The method of claim 1 wherein the second parameter is computed using at predetermined number of data values.

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

14. The method of claim 1 wherein the second parameter is computed by increasing a number of data values until a goodness of fit value is less than a predetermined threshold.

15. The method of claim 1 wherein the second parameter is computed by decreasing a number of data values until a goodness of fit value is greater than a predetermined threshold.

16. The method of claim 1 wherein the offset is a function equal to the first parameter added to the third parameter times an index.

17. The method of claim 16 wherein the index is equal to a cycle number minus one.

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

19. 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.

20. 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.

21. The method of claim 20 wherein the array of reaction chambers comprises a plurality of reaction chambers characterized by a volume of less than or equal to 10 nanoliters.

22. 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.

23. The method of claim 1 wherein the sample comprises a DNA sample, the method further comprising computing a cycle-threshold for the DNA sample using the at least one adjusted amplification curve.

24. A computer-readable medium including computer executable instructions for adjusting amplification curves in a PCR experiment, the computer readable medium including:

one or more instructions for receiving a plurality of amplification curves for a sample;

one or more instructions for computing a first parameter for each of the plurality of amplification curves;

one or more instructions for computing a second parameter for each of the plurality of amplification curves;

one or more instructions for computing a third parameter using at least a portion of the first or second parameters;

one or more instructions for computing an offset for each of the plurality of amplification curves, the offset being a function of the first parameter and the third parameter; and

one or more instructions for adjusting at least one of the plurality of amplification curves by subtracting the offset.

25. A system for characterizing biological processes, the system comprising:

an imaging system adapted to capture a series of images of a microfluidic device, wherein each of the plurality of images includes a plurality of sample chambers;

a processor adapted to;

compute a plurality of amplification curves for each of the plurality of sample chambers;

compute a first parameter for each of the plurality of amplification curves;

compute a second parameter for each of the plurality of amplification curves;

compute a third parameter using at least a portion of the first or second parameters;

compute an offset for each of the plurality of amplification curves, the offset being a function of the first parameter and the third parameter; and

adjust at least one of the plurality of amplification curves by subtracting the offset; and

a memory adapted to store the plurality of adjusted amplification curves.

26. The system of claim 25 wherein the biological processes comprise PCR.

27. The system of claim 25 wherein the series of images comprises a series of fluorescent images.

28. A method for adjusting ratio curves in a PCR experiment, the method comprising:

obtaining a first series of amplification values for a DNA sample;

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

computing 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;

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

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

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

computing a first offset function equal to the first baseline value plus the average slope times an index;

computing a second offset function equal to the second baseline value plus the average slope times the index;

adjusting the first series of amplification values by subtracting the first offset function from the first series of amplification values; and

adjusting the second series of amplification values by subtracting the second offset function from the second series of amplification values.