Statistical combining of cell expression profiles
First Claim
1. A method of fluorophore bias removal comprising the steps of:
- (a) labeling a first pool of genetic matter, derived from a biological system representing a baseline state, with a first fluorophore to obtain a first pool of fluorophore-labeled genetic matter;
(b) labeling a second pool of genetic matter, derived from a biological system representing a perturbed state, with a second fluorophore to obtain a second pool of fluorophore-labeled genetic matter;
(c) labeling a third pool of genetic matter, derived from said biological system representing said baseline state, with said second fluorophore to obtain a third pool of fluorophore-labeled genetic matter;
(d) labeling a fourth pool of genetic matter, derived from said biological system representing said perturbed state, with said first fluorophore to obtain a fourth pool of fluorophore-labeled genetic matter;
(e) contacting said first pool of fluorophore-labeled genetic matter and said second pool of fluorophore-labeled genetic matter with a first microarray under conditions such that hybridization can occur, and determining a first color ratio between said first pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray and said second pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray;
(f) contacting said third pool of fluorophore-labeled genetic matter and said fourth pool of fluorophore-labeled genetic matter with a second microarray under conditions such that hybridization can occur, and determining a second color ratio between said third pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray and said fourth pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray; and
(g) computing an average color ratio by averaging said first color ratio and said second color ratio.
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Abstract
A method for fluorophore bias removal in microarray experiments in which the fluorophores used in microarray experiment pairs are reversed. Further, a method for calculating the individual errors associated with each measurement made in nominally repeated microarray experiments. This error measurement is optionally coupled with rank based methods in order to determine a probability that a cellular constituent is up or down regulated in response to a perturbation. Finally, a method for determining the confidence in the weighted average of the expression level of a cellular constituent in nominally repeated microarray experiments.
31 Citations
42 Claims
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1. A method of fluorophore bias removal comprising the steps of:
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(a) labeling a first pool of genetic matter, derived from a biological system representing a baseline state, with a first fluorophore to obtain a first pool of fluorophore-labeled genetic matter;
(b) labeling a second pool of genetic matter, derived from a biological system representing a perturbed state, with a second fluorophore to obtain a second pool of fluorophore-labeled genetic matter;
(c) labeling a third pool of genetic matter, derived from said biological system representing said baseline state, with said second fluorophore to obtain a third pool of fluorophore-labeled genetic matter;
(d) labeling a fourth pool of genetic matter, derived from said biological system representing said perturbed state, with said first fluorophore to obtain a fourth pool of fluorophore-labeled genetic matter;
(e) contacting said first pool of fluorophore-labeled genetic matter and said second pool of fluorophore-labeled genetic matter with a first microarray under conditions such that hybridization can occur, and determining a first color ratio between said first pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray and said second pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray;
(f) contacting said third pool of fluorophore-labeled genetic matter and said fourth pool of fluorophore-labeled genetic matter with a second microarray under conditions such that hybridization can occur, and determining a second color ratio between said third pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray and said fourth pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray; and
(g) computing an average color ratio by averaging said first color ratio and said second color ratio. - View Dependent Claims (5, 7, 8, 9, 11, 13)
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2. A computer system for fluorophore bias removal, the computer system comprising a processor, and a memory encoding one or more programs coupled to the processor, wherein the one or more programs cause the processor to perform a method comprising:
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(a) labeling a first pool of genetic matter, derived from a biological system representing a baseline state, with a first fluorophore to obtain a first pool of fluorophore-labeled genetic matter;
(b) labeling a second pool of genetic matter, derived from a biological system representing a perturbed state, with a second fluorophore to obtain a second pool of fluorophore-labeled genetic matter;
(c) labeling a third pool of genetic matter, derived from said biological system representing said baseline state, with said second fluorophore to obtain a third pool of fluorophore-labeled genetic matter;
(d) labeling a fourth pool of genetic matter, derived from said biological system representing said perturbed state, with said first fluorophore to obtain a fourth pool of fluorophore-labeled genetic matter;
(e) contacting said first pool of fluorophore-labeled genetic matter and said second pool of fluorophore-labeled genetic matter with a first microarray under conditions such that hybridization can occur, and determining a first color ratio between said first pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray and said second pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray;
(f) contacting said third pool of fluorophore-labeled genetic matter and said fourth pool of fluorophore-labeled genetic matter with a second microarray under conditions such that hybridization can occur, and determining a second color ratio between said third pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray and said fourth pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray; and
(g) computing an average color ratio by averaging said first color ratio and said second color ratio. - View Dependent Claims (6, 10, 12)
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3. A method of fluorophore bias removal, said method comprising determining a color ratio by averaging a first color ratio and a second color ratio wherein said first color ratio and said second color ratio have been determined by:
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(a) labeling a first pool of genetic matter, derived from a biological system representing a baseline state, with a first fluorophore to obtain a first pool of fluorophore-labeled genetic matter;
(b) labeling a second pool of genetic matter, derived from a biological system representing a perturbed state, with a second fluorophore to obtain a second pool of fluorophore-labeled genetic matter;
(c) labeling a third pool of genetic matter, derived from said biological system representing said baseline state, with said second fluorophore to obtain a third pool of fluorophore-labeled genetic matter;
(d) labeling a fourth pool of genetic matter, derived from said biological system representing said perturbed state, with said first fluorophore to obtain a fourth pool of fluorophore-labeled genetic matter;
(e) contacting said first pool of fluorophore-labeled genetic matter and said second pool of fluorophore-labeled genetic matter with a first microarray under conditions such that hybridization can occur, and determining a first color ratio between said first pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray and said second pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray; and
(f) contacting said third pool of fluorophore-labeled genetic matter and said fourth pool of fluorophore-labeled genetic matter with a second microarray under conditions such that hybridization can occur, and determining a second color ratio between said third pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray and said fourth pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray.
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4. A computer system for fluorophore bias removal, the computer system comprising a processor, and a memory encoding one or more programs coupled to the processor, wherein the one or more programs cause the processor to perform a method comprising determining a color ratio by averaging a first color ratio and a second color ratio and said first color ratio and said second color ratio have been determined by:
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(a) labeling a first pool of genetic matter, derived from a biological system representing a baseline state, with a first fluorophore to obtain a first pool of fluorophore-labeled genetic matter;
(b) labeling a second pool of genetic matter, derived from a biological system representing a perturbed state, with a second fluorophore to obtain a second pool of fluorophore-labeled genetic matter;
(c) labeling a third pool of genetic matter, derived from said biological system representing said baseline state, with said second fluorophore to obtain a third pool of fluorophore-labeled genetic matter;
(d) labeling a fourth pool of genetic matter, derived from said biological system representing said perturbed state, with said first fluorophore to obtain a fourth pool of fluorophore-labeled genetic matter;
(e) contacting said first pool of fluorophore-labeled genetic matter and said second pool of fluorophore-labeled genetic matter with a first microarray under conditions such that hybridization can occur, and determining a first color ratio between said first pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray and said second pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray; and
(f) contacting said third pool of fluorophore-labeled genetic matter and said fourth pool of fluorophore-labeled genetic matter with a second microarray under conditions such that hybridization can occur, and determining a second color ratio between said third pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray and said fourth pool of fluorophore-labeled genetic matter that binds under said conditions to said microarray.
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14. A method for determining a probability that an expression level of a cellular constituent in a plurality of paired differential microarray experiments is altered by a perturbation, wherein each paired differential microarray experiment in said plurality of paired differential microarray experiments comprises a first microarray experiment representing a baseline state of a first biological system, and a second microarray experiment representing a perturbed state of said first biological system, said method comprising the steps of
(a) determining an error distribution statistic by fitting a reference pair of microarray experiments with an intensity independent statistic, wherein said reference pair of microarray experiments comprises a first reference microarray experiment, and a second reference microarray experiment that is a nominal repeat of said first reference microarray experiment; -
(b) selecting said cellular constituent from a set of cellular constituents measured in said plurality of paired differential microarray experiments, and, for each paired differential microarray experiment in said plurality of paired differential microarray experiments, determining an amount of change in expression level of said cellular constituent between the second microarray experiment and the first microarray experiment of said paired differential microarray experiment using said error distribution statistic; and
(c) determining said probability that said expression level of said cellular constituent in said plurality of paired differential microarray experiments is altered by said perturbation by combining said amount of change in expression level of said cellular constituent determined in step (b) for each paired differential microarray experiment in said plurality of paired differential microarray experiments using a rank based method. - View Dependent Claims (16, 18, 20, 22, 23, 24, 25, 26, 27)
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15. A computer system for determining a probability that an expression level of a cellular constituent in a plurality of paired differential microarray experiments is altered by a perturbation, wherein each paired differential microarray experiment in said plurality of paired differential microarray experiments comprises a first microarray experiment representing a baseline state of a first biological system, and a second microarray experiment representing a perturbed state of said first biological system;
- the computer system comprising a processor, and a memory encoding one or more programs coupled to the processor and the one or more programs cause the processor to perform a method comprising the steps of
(a) determining an error distribution statistic by fitting a reference pair of microarray experiments with an intensity independent statistic, wherein said reference pair of microarray experiments comprises a first reference microarray experiment, and a second reference microarray experiment that is a nominal repeat of said first reference microarray experiment;
(b) selecting said cellular constituent from a set of cellular constituents measured in said plurality of paired differential microarray experiments, and, for each paired differential microarray experiment in said plurality of paired differential microarray experiments, determining an amount of change in expression level of said cellular constituent between the second microarray experiment and the first microarray experiment of said paired differential microarray experiment using said error distribution statistic; and
(c) determining said probability that said expression level of said cellular constituent in said plurality of paired differential microarray experiments is altered by said perturbation by combining said amount of change in expression level of said cellular constituent determined in step (b) for each paired differential microarray experiment in said plurality of paired differential microarray experiments using a rank based method. - View Dependent Claims (17, 19, 21)
- the computer system comprising a processor, and a memory encoding one or more programs coupled to the processor and the one or more programs cause the processor to perform a method comprising the steps of
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28. A method for determining a weighted mean differential intensity in an expression level of a cellular constituent in a biological system in response to a perturbation, said method comprising:
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(a) determining an error distribution statistic by fitting a reference microarray experiment pair with an intensity independent statistic, wherein said reference microarray experiment pair comprises a first reference microarray experiment, and a second reference microarray experiment that is a nominal repeat of said first reference microarray experiment;
(b) determining an amount of differential expression of said cellular constituent a plurality of times;
(c) for each amount of differential expression determined by step (b), calculating a corresponding amount of error based on a magnitude derived by said error distribution statistic; and
(d) computing said weighted mean differential intensity by inversely weighting each said amount of differential expression of said cellular constituent determined in step (b) by the corresponding amount of error determined in step (c) according to the formula where x is said weighted mean differential intensity in said expression level of said cellular constituent, xi is a measurement of an amount of differential expression of said cellular constituent determined by step (b) and σ
i2 is the corresponding amount of error of xi determined by step (c). - View Dependent Claims (30, 31, 32)
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29. A computer system for determining a weighted mean differential intensity in an expression level of a cellular constituent in a biological system in response to a perturbation bias removal, the computer system comprising a processor, and a memory encoding one or more programs coupled to the processor, wherein the one or more programs cause the processor to perform a method comprising:
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(a) determining an error distribution statistic by fitting a reference microarray experiment pair with an intensity independent statistic, wherein said reference microarray experiment pair comprises a first reference microarray experiment and a second reference microarray experiment which is a nominal repeat of said first reference microarray experiment;
(b) determining an amount of differential expression of said cellular constituent a plurality of times;
(c) for each amount of differential expression determined in accordance with (b), calculating a corresponding amount of error based on a magnitude derived by said error distribution statistic; and
(d) computing said weighted mean differential intensity by inversely weighting each said amount of differential expression of said cellular constituent determined in step (b) by the corresponding amount of error determined in step (c) according to the formula where x is said weighted mean differential intensity of in said expression level of said cellular constituent, xi is an amount of differential expression of said cellular constituent i and σ
i2 is a corresponding error for xi.
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33. A method for determining a confidence of a weighted average of a plurality of cellular constituent differential expression measurements determined for a predetermined cellular constituent, j, wherein each cellular constituent differential expression measurement is determined by a paired differential microarray experiment selected from a plurality of paired differential microarray experiments wherein each paired differential microarray experiment comprises a first microarray experiment representing a baseline state of a biological system and a second microarray experiment representing a perturbed state of a biological system, said method comprising the steps of
(a) determining an error distribution statistic by fitting a reference pair of microarray experiments with an intensity independent statistic, wherein said reference pair of microarray experiments comprises a first reference microarray experiment and a second reference microarray experiment that is a nominal repeat of said first reference microarray experiment; -
(b) for each paired differential microarray experiment in said plurality of paired differential microarray experiments, determining an amount of error based upon said error distribution statistic;
(c) determining a scatter sj for cellular constituent j based upon the plurality of paired differential microarray experiments using a relationship
where xi is a differential measurement of cellular constituent j that is determined by paired differential microarray experiment i, {overscore (X)} is the unweighted mean value of all differential measurements of cellular constituent j in said plurality of paired differential microarray experiments, and N is a number of paired differential microarray experiments in said plurality of paired differential microarray experiments; and
(d) combining said amount of error for each paired differential microarray experiment determined in step (b) with said scatter sj to determine said confidence of said weighted average of said plurality of cellular constituent differential expression measurements determined for said predetermined cellular constituent j. - View Dependent Claims (34, 35, 36, 37)
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38. A computer system for determining a confidence of a weighted average of a plurality of cellular constituent differential expression measurements determined for a predetermined cellular constituent, j, wherein each cellular constituent differential expression measurement is determined by a paired differential microarray experiment selected from a plurality of paired differential microarray experiments wherein each paired differential microarray experiment comprises a first microarray experiment representing a baseline state of a biological system and a second microarray experiment representing a perturbed state of a biological system;
- wherein the computer system comprising a processor, and a memory encoding one or more programs coupled to the processor and the one or more programs cause the processor to perform a method comprising the steps of
(a) determining an error distribution statistic by fitting a reference pair of microarray experiments with an intensity independent statistic, wherein said reference pair of microarray experiments comprises a first reference microarray experiment and a second reference microarray experiment that is a nominal repeat of said first reference microarray experiment;
(b) for each paired differential microarray experiment in said plurality of paired differential microarray experiments, determining an amount of error based upon said error distribution statistic;
(c) determining a scatter sj for cellular constituent j based upon the plurality of paired differential microarray experiments using a relationship
where xi is a differential measurement of cellular constituent j that is determined by paired differential microarray experiment i, {overscore (X)} is the unweighted mean value of all differential measurements of cellular constituent j in said plurality of paired differential microarray experiments, and N is a number of paired differential microarray experiments in said plurality of paired differential microarray experiments; and
(d) combining said amount of error for each paired differential microarray experiment determined in step (b) with said scatter sj to determine said confidence of said weighted average of said plurality of cellular constituent differential expression measurements determined for said predetermined cellular constituent j. - View Dependent Claims (39, 40, 41, 42)
- wherein the computer system comprising a processor, and a memory encoding one or more programs coupled to the processor and the one or more programs cause the processor to perform a method comprising the steps of
Specification