Data processing system and methods
Methods and systems for analysis of image data generated from various reference points. Particularly, the methods and systems provided are useful for real time analysis of image and sequence data generated during DNA sequencing methodologies.
1. A method of identifying a nucleotide base in a nucleic acid sequence, said method comprising:
determining the intensity of each differently colored signal present at a particular feature on an array at a particular time, wherein each differently colored signal corresponds to a different nucleotide base;
repeating said determining step for a plurality of features on an array at said particular time;
refining the signal intensity for said plurality of features then repeating said determining step for said plurality of features; and
selecting for each feature of said plurality of features the refined signal having the highest intensity, wherein said signal having the highest intensity at a particular feature corresponds the identity of the nucleotide base present at said particular feature.
2. A method of selecting a signal from a cluster of signals in an image, comprising:
discarding any signals that do not appear within a cluster of signals, the cluster of signals having a defined cluster radius; and
selecting the signal with the highest intensity of signals within a cluster, wherein the selecting comprises a chastity determination comprising:
(a) generating a list of identified spots and intensities for each template cycle,
(b) providing a preliminary base call for each spot,
(c) determining the chastity of each base call,
(d) identifying spots with two or more chastity failures,
(e) eliminating spots having chastity values lower than a threshold value,
(f) determining the sample diversity and/or the probability that clusters will match base calls by chance, and
(g) analyzing spots in iteration from highest to lowest chastity over template cycles.
3. The method of claim 2 , further comprising discarding any signal which already has a signal neighbor within the cluster radius.
4. The method of claim 2 , wherein said selecting step further comprises ordering the signals of the cluster of signals by intensity or brightness.
5. The method of claim 4 , wherein said intensity or brightness is selected from the group consisting of brightness relative to neighboring pixels, brightness relative to a noise estimate, absolute brightness, brightness as a percentile, and extracted (bilinear) brightness.
6. The method of claim 5 , wherein said brightness is brightness relative to neighboring pixels.
7. The method of claim 6 , wherein said neighboring pixels are present within a defined radius.
8. The method of claim 7 , wherein said defined radius is lowered in response to increased signal density.
9. The method of claim 7 , wherein said defined radius is less than or equal to 2.0 pixels.
10. The method of claim 2 , wherein said selecting step further comprises ordering the signals of the cluster of signals by detection count.
11. The method of claim 10 , wherein said detection count is equivalent to the number of times a signal is detected within a selected radius.
12. The method of claim 11 , wherein said selected radius is measured from a pre-selected signal.
13. The method of claim 12 , wherein said pre-selected signal is the first signal detected within a group of signals.
14. The method of claim 12 , wherein said pre-selected signal is the brightest signal in a predicted feature location.
15. The method of claim 11 , wherein said selected radius is established without reference to a pre-selected signal.
16. The method of claim 11 , wherein said selected radius is about 0.5 pixels.
17. The method of claim 2 , wherein said selecting step further comprises ordering the signals of the cluster by detection count then ordering the signals by intensity, wherein the detection count is equivalent to the number of times a signal is detected within a selected radius.
18. The method of claim 2 , wherein the chastity determination comprises comparing the selected signal with a signal from the cluster of signals having the second highest intensity of signals within the cluster.
19. The method of claim 18 , wherein a first intensity value is divided by the sum of the first intensity value and a second intensity value, wherein the first intensity value is the intensity of the selected signal, and the second intensity value is the intensity of the signal from the cluster of signals having the second highest intensity of signals within the cluster.
20. The method of claim 2 , wherein step (g) comprises:
(i) discarding the spot being analyzed if another spot has been previously assigned within a first radius; and
(ii) discarding the spot being analyzed if another spot has been previously assigned within a second radius and the base calls for the spot meet a threshold chastity value.
21. The method of claim 2 , wherein said first and second radii have different values.
22. The method of claim 2 , wherein said threshold chastity values range from about 0.5 to about 0.99.