Identifying nucleotides by determining phasing
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 system for base calling a nucleotide in a nucleic acid molecule, comprising:
a processor;
a storage device; and
a program comprising instructions for:
acquiring fluorescent emissions from labeled nucleotides hybridized to a cluster of identical nucleic acids bound to a substrate;
calculating the fraction of labeled nucleotides in the cluster that are out of phase;
identifying a particular base called nucleotide based on the acquired fluorescent emissions and the calculated fraction of labeled nucleotides that are out of phase; and
assigning a quality score to the particular base called nucleotide based on the identification.
2. The system of claim 1 , wherein the system comprises a CMOS detector and the program comprises instructions for acquiring fluorescent emissions from images gathered by the CMOS detector.
3. The system of claim 1 , wherein the system comprises a nucleic acid sequencing instrument that performs multiple cycles of sequencing by synthesis.
4. The system of claim 3 , wherein calculating the fraction of labeled nucleotides in the cluster that are out of phase comprises assigning a fixed fraction of the labeled nucleotides in the cluster to be out of phase during each cycle of the multiple cycles of the sequencing by synthesis.
5. The system of claim 3 , wherein calculating the fraction of labeled nucleotides in the cluster that are out of phase comprises calculating the fraction of labeled nucleotides that are out of phase over n cycles.
6. The system of claim 3 , wherein calculating the fraction of labeled nucleotides in the cluster that are out of phase comprises generating a phasing matrix that models the phasing effects at any cycle of the multiple cycles of the sequencing by synthesis.
7. The system of claim 6 , wherein generating the phasing matrix comprises creating an N×N matrix where N is the total number of the multiple cycles of the sequencing by synthesis, each row of the matrix represents cycles and each column of the matrix represents a termination position of a template nucleic acid in the cycle.
8. The system of claim 3 , wherein assigning a quality score comprises generating an output file which contains a quality score for each cycle of the multiple cycles, for the cluster.
9. The system of claim 1 , wherein assigning the quality score to the particular base called nucleotide comprises calculating a set of predictor values for the base call.
10. The system of claim 9 , wherein the program further comprises using the predictor values to look up the quality score in a quality table.
11. The system of claim 9 , wherein the predictor values are selected from the group consisting of: approximate homopolymer; intensity decay; penultimate chastity; signal overlap with background (SOWB) and shifted purity G adjustment.
12. The system of claim 1 , wherein the program comprises a state machine which monitors a file system within the storage device for the presence of a new file.
13. They system of claim 12 , wherein identifying the labeled nucleotides results in generation of a base call file in the storage device.
14. The system of claim 13 , wherein the quality score is calculated from data in the base call file stored on the storage device.
15. The system of claim 1 , further comprising discounting quality scores which are below a predetermined threshold.
16. A method for base calling a nucleotide in a nucleic acid molecule having a particular quality score, comprising:
acquiring fluorescent emissions from labeled nucleotides hybridized to a cluster of identical nucleic acids bound to a substrate, wherein the labeled nucleotides should be the same;
calculating the fraction of labeled nucleotides in the cluster that are out of phase;
identifying the labeled nucleotides based on the acquired fluorescent emissions and the calculated fraction of labeled nucleotides to identify the particular base called nucleotide; and
assigning a quality score to the particular base called nucleotide based on the identification.
17. The method of claim 16 , wherein the method is repeated to perform multiple cycles of sequencing by synthesis.
18. The method of claim 17 , wherein calculating the fraction of labeled nucleotides in the cluster that are out of phase comprises assuming a fixed fraction of the labeled nucleotides in the cluster become out of phase during each cycle of the multiple cycles of the sequencing by synthesis.
19. The method of claim 17 , wherein calculating the fraction p of labeled nucleotides in the cluster that are out of phase, after cycle n, comprises calculating the fraction of molecules that will be phased by k cycles using the formula:
(
n
k
)
(
1
-
p
)
n
-
k
p
k
.
20. The method of claim 17 , wherein calculating the fraction of labeled nucleotides in the cluster that are out of phase comprises generating a phasing matrix to model the phasing effects at any cycle of the multiple cycles of the sequencing by synthesis.
21. The method of claim 16 , wherein the method comprises monitoring a file system to determine when a specimen comprising the nucleotide is ready to advance from one state to the next state in a state machine.
22. They method of claim 21 , wherein identifying the labeled nucleotides results in generation of a base call file.
23. The method of claim 22 , wherein assigning a quality score comprises reading the base call file and calculating a quality score.
24. The method of claim 16 , further comprising discounting quality scores which are below a predetermined threshold.