METHOD AND APPARATUS FOR DETERMINING COPY NUMBER VARIATION USING DIGITAL PCR

• US 20090239308A1
• Filed: 07/09/2008
• Published: 09/24/2009
• Est. Priority Date: 03/19/2008
• Status: Active Grant

• Current Assignee: Standard Biotools Incorporated
• Original Assignee: Fluidigm Corporation
• Inventors: Qin, Jian, Dube, Simant, Ramakrishnan, Ramesh
• Granted Patent: US 9,487,822 B2
• Time in Patent Office: Days
• US Class Current: 436/94
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2537/16 Assays for determining copy number or wherein the copy number is of special importance
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: G01N 35/00594 Quality control, including calibration or testing of components of the analyser
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A method of estimating a concentration of DNA molecules in a biological sample includes storing a number of a plurality of reaction sites in a memory and distributing the biological sample among the plurality of reaction sites. The method also includes determining a number of the plurality of reaction sites characterized by a presence of one or more of the DNA molecules and computing a portion of the plurality of reaction sites characterized by the presence of the one or more of the DNA molecules. The method further includes estimating the concentration of the DNA molecules as a function of the portion of the plurality of reaction sites and computing a confidence interval for the estimated concentration of DNA molecules.

Claims

1. A method of estimating a concentration of DNA molecules in a biological sample, the method comprising:

storing a number of a plurality of reaction sites in a memory;

distributing the biological sample among the plurality of reaction sites;

determining a number of the plurality of reaction sites characterized by a presence of one or more of the DNA molecules;

computing a portion of the plurality of reaction sites characterized by the presence of the one or more of the DNA molecules;

estimating the concentration of the DNA molecules as a function of the portion of the plurality of reaction sites; and

computing a confidence interval for the estimated concentration of DNA molecules.

2. The method of claim 1 wherein computing the confidence interval comprises:

computing a lower bound as a function of the portion of the plurality of reaction sites and a standard deviation; and

computing an upper bound as a function of the portion of the plurality of reaction sites and the standard deviation.

3. The method of claim 1 wherein the biological sample comprises a reference gene.

4. The method of claim 3 wherein the biological sample comprises a target gene different from the reference gene.

5. The method of claim 1 wherein the plurality of reaction sites comprise a plurality of reaction chambers of a nanofluidic chip.

6. The method of claim 5 wherein the plurality of reaction sites comprise at least 765 reaction chambers in fluidic isolation.

7. The method of claim 1 wherein the plurality of reaction sites comprise a plurality of droplets.

8. The method of claim 1 further comprising determining a second portion of the reaction sites characterized by a presence of no DNA molecules.

9. The method of claim 1 wherein estimating the concentration of the DNA molecules comprises multiplying the number of the plurality of reaction sites by the natural logarithm of the quantity one minus the first portion of the plurality of reaction sites.

10. A method of estimating a concentration of a DNA molecule in a biological sample, the method comprising:

storing a number of a plurality of reaction sites in a memory;

distributing the biological sample among the plurality of reaction sites;

determining a number of the plurality of reaction sites characterized by a presence of one or more of the DNA molecules;

computing a portion of the plurality of reaction sites characterized by the presence of the one or more of the DNA molecules;

computing a standard deviation as a function of the portion of the plurality of reaction sites characterized by the presence of the one or more of the DNA molecules;

forming a first probability density function having a mean based on the portion of the plurality of reaction sites characterized by the presence of the one or more of the DNA molecules and the standard deviation;

defining a first limit and a second limit;

computing a first confidence interval associated with an area under the first probability density function between the first limit and the second limit;

forming a second probability density function as a function of a set of concentration values, wherein forming a second probability density function comprises;

computing a hit percentage as a function of a concentration value of the set of concentration values;

computing a second probability density function value as a function of the first probability density function evaluated at the hit percentage; and

repeating computing a first hit percentage and computing a second probability density function for the set of concentration values;

defining a third limit and a fourth limit; and

computing a second confidence interval associated with an area under the second probability density function between the third limit and the fourth limit.

11. The method of claim 10 wherein the first probability density function is defined in terms of a number of positive reaction chambers.

12. The method of claim 10 wherein the second probability density function is defined in terms of estimated concentration values.

13. The method of claim 10 wherein the standard deviation is a function of the square root of the portion of the plurality of reaction sites characterized by the presence of the one or more of the DNA molecules and the number of the plurality of reaction sites.

14. The method of claim 10 wherein the hit percentage is a function of one minus a negative exponential function of a concentration value.

15. The method of claim 10 wherein computing the second probability density function comprises multiplying the first probability density function evaluated at the hit percentage by the quantity one minus the hit percentage.

16. A method of estimating a ratio of a concentration of a first DNA molecule in a biological sample to a concentration of a second DNA molecule in the biological sample, the method comprising:

storing a number of a plurality of reaction sites in a memory;

distributing the biological sample among the plurality of reaction sites;

determining a first number of the plurality of reaction sites characterized by a presence of one or more of the first DNA molecules;

determining a second number of the plurality of reaction sites characterized by a presence of one or more of the second DNA molecules;

computing a first portion of the plurality of reaction sites characterized by the presence of the one or more first DNA molecules;

computing a portion of the plurality of reaction sites characterized by the presence of the one or more second DNA molecules;

estimating the concentration of the first DNA molecule as a function of the first portion of the plurality of reaction sites;

estimating the concentration of the second DNA molecule as a function of the second portion of the plurality of reaction sites; and

computing the ratio of the concentration of the first DNA molecule in the biological sample to the concentration of a second DNA molecule in the biological sample.

17. The method of claim 16 wherein the biological sample comprises a target gene and a reference gene different from the target gene.

18. The method of claim 16 wherein the plurality of reaction sites comprise a plurality of reaction chambers of a nanofluidic chip.

19. The method of claim 18 wherein the plurality of reaction sites comprise at least 765 reaction chambers in fluidic isolation.

20. The method of claim 16 wherein estimating the concentration of the first DNA molecule as a function of the first portion of the plurality of reaction sites comprises multiplying the number of the plurality of reaction sites by the natural logarithm of the quantity one minus the first portion of the plurality of reaction sites.

21. The method of claim 16 wherein estimating the concentration of the second DNA molecule as a function of the second portion of the plurality of reaction sites comprises multiplying the number of the plurality of reaction sites by the natural logarithm of the quantity one minus the second portion of the plurality of reaction sites.

22. A method of estimating a ratio of a concentration of a first DNA molecule in a biological sample to a concentration of a second DNA molecule in the biological sample, the method comprising:

storing a number of analysis panels in a memory;

storing a number of a plurality of reaction sites per analysis panel in the memory;

distributing the biological sample among the plurality of reaction sites of the analysis panels;

forming a first histogram of the concentration of the first DNA molecule per panel based on the number of analysis panels;

forming a second histogram of the concentration of the second DNA molecule per panel based on the number of analysis panels;

forming a ratio histogram, wherein forming the ratio histogram comprises;

defining a bin extent associated with the ratio histogram;

determining a number of ratio pairs from the first histogram and the second histogram, respectively, falling within the bin extent, thereby defining a value associated with the first histogram and a value associated with the second histogram;

computing a product of the first histogram evaluated at the value associated with the first histogram and the second histogram evaluated at the value associated with the second histogram;

storing a histogram entry for the product in the memory associated with the bin extent; and

repeating determining a number of ratio pairs, computing a product, and storing a histogram entry to form the ratio histogram.

23. The method of claim 22 wherein forming a first histogram of the concentration of the first DNA molecule per panel comprises:

determining a number of the plurality of reaction sites per analysis panel characterized by a presence of one or more of the first DNA molecules;

computing a portion of the plurality of reaction sites characterized by the presence of the one or more of the first DNA molecules;

computing a standard deviation as a function of the portion of the plurality of reaction sites characterized by the presence of the one or more of the first DNA molecules; and

forming a first probability density function having a mean based on the portion of the plurality of reaction sites characterized by the presence of the one or more of the first DNA molecules and the standard deviation.

24. The method of claim 23 further comprising:

defining a first limit and a second limit; and

computing a first confidence interval associated with an area under the first probability density function between the first limit and the second limit.

25. The method of claim 22 wherein forming a second histogram of the concentration of the second DNA molecule per panel comprises:

determining a number of the plurality of reaction sites per analysis panel characterized by a presence of one or more of the second DNA molecules;

computing a portion of the plurality of reaction sites characterized by the presence of the one or more of the second DNA molecules;

computing a standard deviation as a function of the portion of the plurality of reaction sites characterized by the presence of the one or more of the second DNA molecules; and

forming a second probability density function having a mean based on the portion of the plurality of reaction sites characterized by the presence of the one or more of the second DNA molecules and the standard deviation.

26. The method of claim 25 further comprising:

defining a first limit and a second limit; and

computing a first confidence interval associated with an area under the first probability density function between the first limit and the second limit.

27. A system for estimating a concentration of a DNA molecule in a biological sample, the systems comprising:

a light source;

a first optical system in optical communication with the light source;

a nanofluidic chip support configured to receive a nanofluidic chip having a plurality of reaction chambers, wherein optical radiation from the light source is directed onto the nanofluidic chip by the first optical system;

a second optical system configured to receive optical radiation emitted from the nanofluidic chip;

a detector in optical communication with the second optical system; and

a computer comprising a data processor and a computer-readable medium storing a plurality of instructions for controlling a data processor to estimate the concentration of the DNA molecule in the biological sample, the plurality of instructions comprising;

instructions that cause the data processor to determine a number of the plurality of reaction chambers characterized by a presence of one or more of the DNA molecules;

instructions that cause the data processor to compute a portion of the plurality of reaction sites characterized by the presence of the one or more of the DNA molecules;

instructions that cause the data processor to estimate the concentration of the DNA molecules as a function of the portion of the plurality of reaction sites; and

instructions that cause the data processor to compute a confidence interval for the estimated concentration of DNA molecules.