Imaging microarrays

• US 7,636,636 B2
• Filed: 10/11/2002
• Issued: 12/22/2009
• Est. Priority Date: 10/12/2001
• Status: Expired due to Fees

• Current Assignee: Abbott Molecular Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: Piper, James R.
• Primary Examiner(s): SMITH, CAROLYN L
• Application Number:
  US10/269,723
• Publication Number:
  US 20030124589A1
• Time in Patent Office: 2,629 Days
• Field of Search: 702/19, 702/22
• US Class Current: 702/19
• CPC Class Codes: G06T 2207/10056 Microscopic image
• CPC Class Codes: G06T 2207/10064 Fluorescence image
• CPC Class Codes: G06T 2207/30072 Microarray; Biochip, DNA array; Well plate
• CPC Class Codes: G06T 5/70 Denoising; Smoothing
• CPC Class Codes: G06T 7/0012 Biomedical image inspection
• CPC Class Codes: G06T 7/11 Region-based segmentation
• CPC Class Codes: G06T 7/143 involving probabilistic approaches, e.g. Markov random field [MRF] modelling
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

Abstract

A method of obtaining a corrected image of a microarray includes acquiring an image of a microarray including a target spot, and processing the image to correct for background noise and chip misalignment. The method also includes analyzing the image to identify a target patch, edit debris, and correct for ratio bias; and detecting single copy number variation in the target spot using an objective statistical analysis that includes a t-value statistical analysis. The method provides statistically robust computational processes for accurately detecting genomic variation at the single copy level.

Claims

1. A method of detecting single copy number variation in a target spot of a genomic microarray comprising:

acquiring an image of said microarray including said target spot;

processing the image to correct for chip misalignment;

processing the image to correct for background noise;

analyzing the image to identify a target patch;

analyzing the image to edit debris;

analyzing the image to correct for ratio bias;

detecting single copy number variation in the target spot by an objective statistical analysis;

wherein the genomic microarray includes a test genomic material marked with receptors having a first wavelength and a reference genomic material marked with receptors having a second wavelength, the test and reference genomic materials forming the target spot by a hybridization process;

further comprising measuring a fluorescent signal intensity of the target spot from the test genomic material and the reference genomic material, wherein the fluorescent signal intensity of the first wavelength is proportional to a copy number of the test genomic material and the fluorescent signal intensity of the second wavelength is proportional to a copy number of the reference genomic material;

obtaining an image of a theoretical set of patches; and

,

cross-correlating the image of the theoretical set of patches to the image of the microarray.

2. The method of claim 1, wherein the target spot is Deoxyribonucleic Acid (DNA).

3. The method of claim 1, further comprising preparing a genomic material prior to acquiring the image and generating an array of the genomic material on a solid substrate.

4. The method of claim 1, wherein the genomic material includes a range between 50 and 200 kbp.

5. The method of claim 3, wherein preparing the genomic material includes isolating the genomic material by an extraction process followed by nick translation labeling and polymerase chain reaction (PCR) labeling.

6. The method of claim 1, wherein the hybridization process is a comparative genomic hybridization (CGH) process.

7. The method of claim 1, wherein subsequent to acquiring the image of the genomic microarray and prior to correcting for the background noise, processing the image includes automatically detecting misalignment of the genomic microarray and correcting for rotation of the genomic microarray.

13. The method of claim 1, wherein editing of debris includes automatically removing spot debris by recognizing and excluding spot pixels within a target spot having outlying intensity relative to a majority of the spot pixels.

14. The method of claim 13, wherein editing of debris includes:

transforming test and reference intensities to polar coordinates having as a first coordinate the overall intensity of spot signals and having as a second coordinate the ratio;

applying a standard statistical test for outliers to the first and second coordinates; and

,

analyzing the shape of excluded spot pixels.

8. A method of detecting single copy number variation in a target spot of a genormic microarray comprising:

acquiring an image of said microarray including said target spot;

processing the image to correct for chip misalignment;

processing the image to correct for background noise;

analyzing the image to identify a target patch;

analyzing the image to edit debris;

analyzing the image to correct for ratio bias;

detecting single copy number variation in the target spot by an objective statistical analysis;

wherein the genomic microarray includes a test genomic material marked with receptors having a first wavelength and a reference genomic material marked with receptors having a second wavelength, the test and reference genomic materials forming the target spot by a hybridization process;

further comprising measuring a fluorescent signal intensity of the target spot from the test genomic material and the reference genomic material, wherein the fluorescent signal intensity of the first wavelength is proportional to a copy number of the test genomic material and the fluorescent signal intensity of the second wavelength is proportional to a copy number of the reference genomic material;

wherein correcting of the background noise includes;

computing the acquired image;

computing a minimum image based on the computed acquired image;

computing a maximum image based on the computed minimum image;

subtracting a background image from the computed acquired image to obtain a resulting image; and

,

optimizing the correction for the background noise by identifying a background peak of the resulting image and subtracting the resulting image to obtain a mean value of zero for a corrected image.

9. The method of claim 8, wherein the corrected image includes pixels having signed values.

10. The method of claim 8, wherein identifying a target patch includes:

obtaining an image of a theoretical set of patches; and

,

cross-correlating the image of the theoretical set of patches to the image of the microarray.

11. The method of claim 9, wherein following identifying a target patch, the method further comprises:

computing a threshold by analyzing a pixel intensity of the counterstained image to determine an initial segmentation of the target spot; and

,

performing a process for spot shape analysis and spot segmentation.

12. The method of claim 11, further comprising:

performing a spot identification analysis; and

performing an artifact spot exclusion process wherein an artifact spot is automatically excluded.

15. A method of detecting single copy number variation in a target spot of a genomic microarray comprising:

acquiring an image of said microarray including said target spot;

processing the image to correct for chip misalignment;

processing the image to correct for background noise;

analyzing the image to identify a target patch;

analyzing the image to edit debris;

analyzing the image to correct for ratio bias;

detecting single copy number variation in the target spot by an objective statistical analysis;

wherein the genomic microarray includes a test genomic material marked with receptors having a first wavelength and a reference genomic material marked with receptors having a second wavelength, the test and reference genomic materials forming the target spot by a hybridization process;

further comprising a fluorescent signal intensity of the target spot from the test genomic material and the reference genomic material, wherein the fluorescent signal intensity of the first wavelength is proportional to a copy number of the test genomic material and the fluorescent signal intensity of the second wavelength is proportional to a copy number of the reference genomic material;

further comprising;

computing a ratio based on a test and a reference fluorescence signal intensity value associated with the target spot;

further wherein correcting for ratio bias includes measuring a raw ratio representing a ratio of total test fluorescence intensity from the target spot and a total reference fluorescence intensity from the target spot;

normalizing the raw ratio by dividing the raw ratio of each spot by the median over all spots of the raw ratio; and

further wherein correcting for ratio bias includes;

selecting a subset of spots that have a normalized observed ratio approximately equal to 1.0, referred to as “

plausibly modal spots”

;

modeling an effect of bias in said subset of plausibly modal spots by the equation R=r<sup>−

B</sup>, where R represents said normalized observed ratio, r is the reference intensity of the spot, and B is a bias parameter that is constant across all spots of a microarray;

estimating the optimum value of B from said subset of plausibly modal spots;

performing ratio bias correction of unknown data by applying the correction formula R_corrected=R_original/R_predicted and choosing a value of B that minimizes the mean square difference between the set of normalized original ratios, and the set of normalized predicted ratios according to equation R=r<sup>−

B</sup>.

16. A method of detecting single copy number variation in a target spot of a genomic microarray comprising:

acquiring an image of said microarray including said target spot;

processing the image to correct for chip misalignment;

processing the image to correct for background noise;

analyzing to identify a target patch;

analyzing the image to edit debris;

analyzing the image to correct for ratio bias;

detecting single copy number variation in the target spot by an objective statistical analysis;

wherein the genomic microarray includes a test genomic material marked with receptors having a first wavelength and a reference receptors having a second wavelength, the test and reference genomic materials forming the target spot by a hybridization process;

further comprising measuring a fluorescent signal intensity of the target spot from the test genomic material and the reference genomic material, wherein the fluorescent signal intensity of the first wavelength is proportional to a copy number of the test genomic material and the fluorescent signal intensity of the second wavelength is proportional to a copy number of the reference genomic material;

wherein the objective statistical analysis includes a target-modal statistical analysis; and

wherein the target-modal statistical analysis includes;

for a selected subset of plausibly modal targets, estimating a statistical distribution of ratios of plausibly modal targets; and

for each target of the microarray, computing an estimated target mean and a lower 95% confidence interval limit; and

for each target, reducing the lower 95% confidence interval limit by the median 95% confidence interval over all targets of the microarray; and

for each target, calculating a confidence estimate.

17. The method of claim 16, further comprising estimating a relative copy number of a genomic sequence from the target spot by the target-modal statistical analysis.

18. A computer program product residing on a computer storage medium having instructions stored thereon which, when executed by a logic processor, cause the processor to:

acquire an image of a microarray including a target spot;

process the image to correct for background noise and chip misalignment;

analyze the image to detect target spots;

analyze the image to identify the target patch, edit debris, and correct for ratio bias;

detect single copy number variation in the target spot by an objective statistical analysis;

wherein genomic microarray includes a test genomic material marked with receptors having a first wavelength and a reference genomic material marked with receptors having a second wavelength, the test and reference genomic materials forming the target spot by a hybridization process;

measure a fluorescent signal intensity of the target spot from the test genomic material and the reference genomic material, wherein the fluorescent signal intensity of the first wavelength is proportional to a copy number of the test genomic material and the fluorescent signal intensity of the second wavelength is proportional to a copy number of the reference genomic material;

obtain an image of a theoretical set of patches; and

cross-correlate the image of the theoretical set of patches to the image of the microarray.

19. The computer program product of claim 18, wherein the target spot is Deoxyribonucleic Acid (DNA).

20. The computer program product of claim 18, wherein subsequent to causing the processor to acquire the image of the genomic microarray and prior to causing the processor to process the image for correcting the background noise, causing the processor to process the image further includes automatically causing the processor to detect misalignment of the genomic microarray and correct rotation of the genomic microarray.

21. The computer program product of claim 18, wherein the genomic material includes a range between 50 kbp and 200 kbp.

22. The computer program product of claim 18, wherein the genomic microarray includes a test genomic material marked with a first label having a first wavelength and a reference genomic material marked with a second label having a second wavelength, both test and reference genomic materials forming the target spot by a hybridization process.

23. The computer program product of claim 18, further causing the processor to:

compute a ratio based on a test and a reference fluorescence signal intensity value associated with the target spot.

24. The computer program product of claim 20, wherein correcting for ratio bias includes measuring a raw ratio representing a ratio of total test fluorescence intensity from the test fluorescence signal intensity value and a total reference fluorescence intensity from the reference fluorescence signal intensity value.

25. The computer program product of claim 24, further causing the processor to normalize the raw ratio by mathematical computation.

26. The computer program product of claim 25, wherein correcting for ratio bias includes:

selecting a subset of spots that have a normalized observed ratio approximately equal to 1.0, referred to as “

plausibly modal spots”

;

modeling an effect of bias in said subset of plausibly modal spots by the equation R=r<sup>−

B</sup>, where R represents said normalized observed ratio, r is the reference intensity of the spot, and B is a bias parameter that is constant across all spots of a microarray;

estimating the optimum value of B from said subset of plausibly modal spots;

performing ratio bias correction of unknown data by applying the correction formula R_corrected=R_original/R_predicted and choosing a value of B that minimizes the mean square difference between the set of normalized original ratios, and the set of normalized predicted ratios according to equation R=r<sup>−

B</sup>.

27. The computer program product of claim 18, wherein the objective statistical analysis includes a target-modal statistical analysis.

28. The computer program product of claim 27, wherein the target-modal statistical analysis includes:

for a selected subset of plausibly modal targets, estimating a statistical distribution of ratios of plausibly modal targets; and

for each target of the microarray, computing an estimated target mean and a lower 95% confidence interval limit; and

for each target, modifying the lower 95% confidence interval limit by the median 95% confidence interval over all targets of the microarray; and

for each target, calculating a confidence estimate for the target by comparing the adjusted confidence interval limit with the distribution of ratios of plausibly modal targets.

29. A processor and a memory configured to:

acquire an image of a microarray including at least one target spot;

process the image to correct for background noise and chip misalignment;

analyze the image to detect target spots;

analyze the image to identify a target patch, edit debris, and correct for ratio bias; and

,

detect single copy number variation in a target spot by an objective statistical analysis;

wherein the microarray includes a test genomic material marked with receptors having a first wavelength and a reference genomic material marked with receptors having a second wavelength, the test and reference genomic materials forming the target spot by a hybridization process;

measure a fluorescent signal intensity of the target spot from the test genomic material and the reference genomic material, wherein the fluorescent signal intensity of the first wavelength is proportional to a copy number of the test genomic material and the fluorescent signal intensity of the second wavelength is proportional to a copy number of the reference genomic material;

obtain an image of a theoretical set of patches; and

,

cross-correlate the image of the theoretical set of patches to the image of the microarray.

30. A system comprising:

means for acquiring an image of a microarray including at least one target spot;

means for processing the image to correct for background noise and chip misalignment;

means to analyze the image to detect target spots;

means for analyzing the image to identify a target patch, edit debris, and correct for ratio bias; and

,

means for detecting single copy number variation in the target spot by an objective statistical analysis;

wherein the microarray includes a test genomic material marked with receptors having a first wavelength and a reference genomic material marked with receptors having a second wavelength, the test and reference genomic materials forming the target spot by a hybridization process;

means for measuring a fluorescent signal intensity of the target spot from the test genomic material and the reference genomic material, wherein the fluorescent signal intensity of the first wavelength is proportional to a copy number of the test genomic material and the fluorescent signal intensity of the second wavelength is proportional to a copy number of the reference genomic material;

means for obtaining an image of a theoretical set of patches; and

,

means for cross-correlating the image of the theoretical set of patches to the image of the microarray.