Systems and methods for evaluating the significance of differences in biological measurements
AssigneeRosetta Inpharmatics LLC
InventorsRoland Stoughton, Hongyue Dai
Life of this patent
Estimating the term…
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.
Claims53
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.
2-42. (canceled)
A method of estimating a significance d in a difference between a measurement of a cellular constituent in a first microarray experiment and a measurement of the cellular constituent in a second microarray experiment comprising computing:
- d
- =
- (
- X
- -
- Y
- )
- σ
- X
- 2
- +
- σ
- Y
- 2
- +
- f
- 2
-
- (
- X
- 2
- +
- Y
- 2
- )
- wherein
- X is a brightness of a probe spot representing said cellular constituent in said first microarray experiment;
- Y is a brightness of a probe spot representing said cellular constituent in said second microarray experiment;
- σX2 is a variance term for X that represents an additive error level in X;
- σY2 is a variance term for Y that represents an additive error level in Y;
- f is a fractional multiplicative error level;
- σX2+f2X2 is an estimated variance for X;
- and
- σY2+f2Y2 is an estimated variance for Y.
A method of estimating a significance d in a difference between a measurement of a cellular constituent in a first microarray experiment and a measurement of the cellular constituent in a second microarray experiment comprising computing:
- d
- =
- (
- X
- -
- Y
- )
- σ
- X
- -
- Y
- wherein
- X is a brightness of a probe spot representing said cellular constituent in said first microarray experiment;
- Y is a brightness of a probe spot representing said cellular constituent in said second microarray experiment;
- and
- σX-Y is the combined error in the measurement of said cellular constituent in said first microarray experiment and the measurement of said cellular constituent in said second microarray experiment.
A method of estimating a significance d in a difference between a measurement of a cellular constituent in a plurality of first microarray experiments and a measurement of the cellular constituent in a plurality of second microarray experiments comprising computing:
- d
- =
- (
- x
- -
- y
- )
- σ
- x
- -
- y
- wherein
- the brightness of the probe spot X representing said cellular constituent in the plurality of first microarray experiments are combined to form the weighted mean x;
- the brightness of the probe spot Y representing said cellular constituent in the plurality of second microarray experiments are combined to form the weighted mean y;
- σx-y is the combined error in the measurement of said cellular constituent in the plurality of first microarray experiments and the measurement of said cellular constituent in the plurality of second microarray experiments;
- wherein
- σ
- x
- -
- y
- =
- σ
- x
- 2
- +
- σ
- y
- 2
- ;
- x
- =
- ∑
- x
- i
- σ
- xi
- 2
- ∑
- (
- 1
- σ
- xi
- 2
- )
-
-
- and
-
-
- y
- =
- ∑
- y
- i
- σ
- yi
- 2
- ∑
- (
- 1
- σ
- yi
- 2
- )
- ;
- xi is the observed brightness of probe spot X in a microarray experiment i in the plurality of first microarray experiments;
- yi is the observed brightness of probe spot Y in a microarray experiment i in the plurality of second microarray experiments;
- σxi2 is the estimated variance in xi in a microarray experiment i in the plurality of first microarray experiments;
- and
- σyi2 is the estimated variance in yi in a microarray experiment i in the plurality of second microarray experiments;
- and wherein when each of said plurality of first microarray experiments and said plurality of second microarray experiments approaches one
- σx approaches
- 1
- ∑
- (
- 1
- σ
- xi
- 2
- )
- ,
- and σy approaches
- 1
- ∑
- (
- 1
- σ
- yi
- 2
- )
- ,
- and wherein when each of said plurality of first microarray experiments and said plurality of second microarray experiments approaches a large number
- σx approaches observed error from scatter in X, and
- σy approaches observed error from scatter in Y.
A method of determining an error σx of a weighted mean x for a quantitative measurement of a cellular constituent, the method comprising:
- obtaining a plurality of instances N of said quantitative measurement, wherein each instance xi of said quantitative measurement is from a first microarray experiment or a nominal repeat of said first microarray experiment;
- computing
- x
- =
- ∑
- x
- i
- σ
- i
- 2
- ∑
- (
- 1
- σ
- i
- 2
- )
- wherein
- x is the weighted mean of the quantitative measurement;
- σi2 is the estimated variance of instance xi of said quantitative measurement;
- and
- computing said error in the mean such that,
- when N approaches 1,
- σx approaches
- 1
- ∑
- (
- 1
- σ
- i
- 2
- )
- and when N approaches a large number of nominal repeats σx approaches observed error from scatter in x.
Dependencies are inferred from the claim text.