Image analysis of high-density synthetic DNA microarrays
First Claim
1. A method for evaluating an image of a hybridized microarray, comprising:
- obtaining an image of a microarray having a plurality of individual probe cells;
estimating the background intensity in the image based on a multivariate statistical model comprising at least one of;
(a) a blurring kernel used to deconvolute blur in the image; and
(b) a multivariate statistical spatial model for the background; and
determining estimated intensity values of pixels in the image by based on data from the background estimation.
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Abstract
Methods, systems, and computer program products for analyzing images of high density microarray chips analyze the image by estimating background using a blurring kernel and/or a spatial multivariate statistical model of the background. The methods, systems, and computer program products can employ a multivariate statistical model and/or a blurring kernel to obtain more representative hybridization intensity results, particularly for pixels in boundary regions of the probe cells. The methods allow for alternative microarray configurations of nucleic acid probes and do not require the use of mismatch probes and can be independent of the type of nucleotide sequence used. Associated microarrays and systems are also described.
40 Citations
43 Claims
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1. A method for evaluating an image of a hybridized microarray, comprising:
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obtaining an image of a microarray having a plurality of individual probe cells;
estimating the background intensity in the image based on a multivariate statistical model comprising at least one of;
(a) a blurring kernel used to deconvolute blur in the image; and
(b) a multivariate statistical spatial model for the background; and
determining estimated intensity values of pixels in the image by based on data from the background estimation. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method for evaluating an image of an expressed microarray, comprising:
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obtaining an image of an expressed microarray having a plurality of individual probe cells;
estimating the locations of each probe cell undergoing analysis, each probe cell location being and regions proximate thereto-defining pixels influenced by the fluorescence or lack of fluorescence of the probe cell;
determining first estimated pixel intensity values for pixels in the probe cell locations;
estimating the intensity of the background for pixels in the image to estimate a spatial distribution of the background intensity in the image; and
for each pixel in the image, reducing the first estimated pixel intensity value to a second estimated pixel intensity value based on the data provided by the estimated intensity of background. - View Dependent Claims (14, 15, 16, 17)
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18. A method for evaluating an image of an HSDM, comprising:
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obtaining an image of an HSDM having a plurality of individual probe cells;
estimating the location of each probe cell undergoing analysis in the image, the probe cell location including a plurality of pixels;
obtaining first estimated pixel intensity values for pixels in a region associated with the probe cell in the image;
estimating background intensity for each probe cell region to obtain a spatial distribution of the background intensity in the image, wherein the step of estimating is performed such that the background intensity of the pixels can vary pixel to pixel in the image; and
determining second estimated pixel intensity values for each probe cell by reducing the first estimated pixel intensity value by its corresponding estimated background intensity. - View Dependent Claims (19, 20, 21, 22, 23)
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24. A method of analyzing data obtained from an image of an hybridized microarray, comprising:
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analyzing image data by using a blurring kernel to deconvolute the blurred probe cells in the image to more closely represent the intensity of the fluorescence over the entire probe cell; and
generating a revised image with adjusted pixel intensity values based on the analysis of the image data. - View Dependent Claims (25, 26, 27)
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28. A computer program product for analyzing an image of a hybridized nucleic acid microarray chip, the computer program product comprising:
a computer readable storage medium having computer readable program code embodied in said medium, said computer-readable program code comprising;
computer readable program code that obtains data of an image of the intensities of a hybridized microarray having a plurality of individual probe cells;
computer readable program code that determines first estimated intensity values of pixels in the image;
computer readable program code that calculates estimates the intensity of background for pixels in the image based on at least one of;
(a) a multivariate statistical spatial model of the background; and
(b) a blurring kernel to deconvolute the blurring in the image; and
computer readable program code that determines second estimated intensity values of pixels in the image by correcting the first estimated values based on data obtained by the estimated intensity of background. - View Dependent Claims (29, 30, 31, 32, 33, 34)
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35. A computer program product for analyzing data representing an image of a hybridized nucleic acid microarray chip, the computer program product comprising:
a computer readable storage medium having computer readable program code embodied in said medium, said computer-readable program code comprising;
computer readable program code that obtains intensity data of an image of a hybridized microarray having a plurality of probe cells; and
computer readable program code that calculates an estimated spatial distribution of the intensity of the background in the image, the estimated intensity of the background being determined based on pixels in the image which correspond to locations on the array which have active nucleic acid probes.
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36. A system for analyzing images of hybridized arrays of nucleic acid probes, comprising:
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a processor; and
means for estimating background in an image using a predetermined spatial multivariate statistical model for the background.
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37. A microarray having a substrate and a plurality of nucleic acid probe cells positioned on a primary surface thereof, wherein said probe cells have a hexagonal shaped perimeter.
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38. An array of oligonucleotide probes immobilized on a solid support, wherein said array has a hybridization surface which is substantially free of mismatch probes.
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39. An array of oligonucleotide probes immobilized on a solid support, wherein said array is sized at about 1.28 cm×
- 1.28 cm or less, and wherein said array comprises at least about 400,000 individual perfect match probe cells thereon.
- View Dependent Claims (40)
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41. An array of oligonucleotide probes immobilized on a solid support, wherein said array has a hybridization surface which is substantially free of mismatch probes, and wherein said probes are sized to cover an area on the hybridization surface which is about 21.5 μ
- m×
21.5 μ
m or less. - View Dependent Claims (42)
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43. A method of classifying the results of hybridization in expression probe arrays of nucleic acid probes, comprising:
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estimating the background intensity in an image of a hybridized microarray using at least one of a blurring kernel to deconvolute blur in the image and a spatial multivariate model for the background;
adjusting the image intensity based on data provided by said estimating step;
ranking the probe cells based on the adjusted intensity, wherein said step of ranking is carried out without regard to information from mismatch probes; and
classifying the results of the hybridization based on said step of ranking.
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Specification