Methods and systems for analyzing image data

Patent metadata

Field	Value
Publication number	US-10689696-B2
Application number	US-201415153953-A
Country	US
Kind code	B2
Filing date	Dec 3, 2014
Priority date	Dec 3, 2013
Publication date	Jun 23, 2020
Grant date	Jun 23, 2020

Abstract

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.

First claim

What is claimed is:

1. A method comprising:
(a) performing a plurality of cycles of a sequencing by synthesis reaction such that, at each cycle, a signal is generated that is indicative of incorporation of a same nucleotide into a plurality of identical polynucleotides, whereby a portion of the signal is noise associated with phasing or pre-phasing;
(b) detecting the signal at each cycle in at least one channel, wherein the signal at each cycle includes an intensity value for each channel; and
(c) preforming cycle-by-cycle phasing corrections by applying a new first order phasing correction at each cycle to the intensity value for each channel;
wherein the new first order phasing correction is calculated for each channel of each cycle, wherein the new first order phasing correction includes subtracting an intensity value of an immediately previous cycle from an intensity value of a current cycle and also includes subtracting an intensity value of an immediately subsequent cycle from the intensity value of the current cycle; and
wherein the same nucleotide incorporated into the plurality of identical polynucleotides is identified for each cycle.

2. The method of claim 1 , wherein the at least one channel includes first and second channels and the same nucleotide is identified at each cycle through two-channel base calling that is performed by extracting image data from only two channels.

3. The method of claim 1 , wherein the new first order phasing correction includes phasing and pre-phasing weights and the method further comprises selecting the phasing and pre-phasing weights from a plurality of possible phasing and pre-phasing weights.

4. The method of claim 3 , wherein the phasing and pre-phasing weights are selected using a pattern search over the possible phasing and pre-phasing weights.

5. The method of claim 1 , wherein detecting the singal at each cycle in the at least one channel includes detecting the singal in only one channel.

6. The method of claim 1 , wherein the signal is detected from a first channel, a second channel, a third channel, and a fourth channel.

7. The method of claim 1 , wherein optimal phasing and pre-phasing weights of the new first order phasing correction are chosen for each cycle by iterating over different phasing corrections calculated with different phasing and pre-phasing weights.

8. The method of claim 1 , wherein phasing and pre-phasing weights of the new first order phasing correction are chosen to optimize a chastity determination for the current cycle.

9. The method of claim 8 , wherein the chastity determination is a mean chastity for the current cycle.

10. A system comprising:
a processor;
a storage capacity; and
a program comprising instructions for:
(a) performing a plurality of cycles of a sequencing by synthesis reaction such that, at each cycle, a signal is generated that is indicative of incorporation of a same nucleotide into a plurality of identical polynucleotides, whereby a portion of the signal is noise associated with phasing or pre-phasing;
(b) detecting the signal at each cycle in at least one channel, wherein the signal at each cycle includes an intensity value for each channel; and
(c) performing cycle-by-cycle phasing corrections by applying a new first order phasing correction at each cycle to the intensity value for each channel;
wherein the new first order phasing correction is calculated for each channel at each cycle, wherein the new first order phasing correction includes subtracting an intensity value of an immediately previous cycle from an intensity value of a current cycle and also includes subtracting an intensity value of an immediately subsequent cycle from the intensity value of the current cycle; and
wherein the same nucleotide incorporated into the plurality of identical polynucleotides is identified for each cycle.

11. The system of claim 10 , wherein the new first order phasing correction includes phasing and pre-phasing weights and the program includes instructions for selecting the phasing and pre-phasing weights from a plurality of possible phasing and pre-phasing weights.

12. The system of claim 10 , wherein the signal is detected from only one channel.

13. The system of claim 10 , wherein the signal is detected from a first channel, a second channel, a third channel, and a fourth channel.

14. The system of claim 10 , wherein the program includes instructions for choosing optimal phasing and pre-phasing weights of the new first order phasing correction for each cycle by iterating over different phasing corrections calculated with different phasing and pre-phasing weights.

15. The system of claim 10 , wherein the program includes instructions for choosing phasing and pre-phasing weights of the new first order phasing correction to optimize a chastity determination for the current cycle.

16. The system of claim 15 , wherein the chastity determination is a mean chastity for the current cycle.

17. A method comprising:
(a) performing a plurality of cycles of a sequencing by synthesis reaction such that, at each cycle, a signal is generated that is indicative of incorporation of a same nucleotide into a plurality of identical polynucleotides, whereby a portion of the signal is noise associated with phasing or pre-phasing;
(b) detecting the signal at each cycle, wherein detecting the signal at each cycle includes detecting an intensity value in a first channel and detecting an intensity value in a second channel; and
(c) performing cycle-by-cycle phasing corrections by applying a new first order phasing correction to the intensity value in each of the first and second channels at each cycle;
wherein the new first order phasing correction is calculated for each of the first and second channels at each cycle, wherein the new first order phasing correction includes subtracting an intensity value of an immediately previous cycle from an intensity value of a current cycle and also includes subtracting an intensity value of an immediately subsequent cycle from the intensity value of a current cycle;
wherein the same nucleotide incorporated into the plurality of identical polynucleotides is identified, for each cycle, based on a combination of the intensity values in the first and second channels.

18. The method of claim 17 , wherein a first nucleotide is detected in the first channel, a second nucleotide is detected in the second channel, a third nucleotide is detected in both the first and the second channels, and a fourth nucleotide is not detected or is minimally detected by the first and second channels.

19. The method of claim 17 , wherein the same nucleotide is one of four nucleotides and is identified through two-channel base calling at each cycle that includes fitting four Gaussian distributions to a data set of two-channel intensity data such that one Gaussian distribution is applied for each of the four nucleotides represented in the data set.

20. The method of claim 19 , wherein the Gaussian distributions are fit to the data set using a clustering algorithm.

21. The method of claim 20 , wherein the plurality of identical polynucleotides forms a cluster that generates the signal and wherein performing the plurality of cycles inclues generating respective signals from multiple clusters, each of the clusters having a different plurality of identical nucleotides, each of the clusters having a pair of X, Y intensity values, X and Y referring to the first and second channels, respectively, wherein for each pair

Assignees

• Illumina Inc

Inventors

• Belitz Paul
• Tanner Stephen
• Vieceli John S
• Chen Xiaoyu

Classifications

• G16B30/00

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• C12Q1/6869 Primary

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• C12Q2537/149

  Sequential reactions · CPC title

• Y02A90/10

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