Determining and removing inter-cluster light interference

Abstract

This disclosure describes embodiments of methods, systems, and non-transitory computer readable media that accurately estimates the crosstalk from an adjacent cluster of oligonucleotides onto a target cluster of oligonucleotides and removes or reduces the crosstalk emitted by the adjacent cluster of oligonucleotides from the target cluster of oligonucleotides. For instance, the disclosed systems can detect the intensity values for a target cluster and the adjacent cluster. Based on the intensity values of the adjacent cluster, the disclosed systems can determine an inter-cluster-interference metric that estimates the crosstalk emitted from the adjacent cluster. The disclosed systems can remove the inter-cluster-interference metric from the intensity value of the target cluster and generate modified intensity values for the target cluster.

Description

Abstract This disclosure describes embodiments of methods, systems, and non-transient computer-readable media that can accurately estimate crosstalk between neighboring oligonucleotide clusters and a target oligonucleotide cluster, and eliminate or reduce such crosstalk. For example, the disclosed system can detect the intensity values ​​of a target cluster and neighboring clusters. Based on the intensity values ​​of neighboring clusters, the disclosed system can determine an inter-cluster interference metric that estimates the crosstalk emitted by neighboring clusters. The disclosed system can remove the inter-cluster interference metric from the intensity value of the target cluster, producing a corrected intensity value for the target cluster.

Claims

CLAIMSWhat is claimed is:

1. A system comprising: at least one processor; and a non-transitory computer readable medium comprising instructions that, when executed by the at least one processor, cause the system to: detect, for a sequencing cycle, a first set of intensity values for a first signal from a first cluster of oligonucleotides and a second set of intensity values for a second signal from a second cluster of oligonucleotides; determine a set of illumination indicators representing whether the first cluster of oligonucleotides is illuminated during the sequencing cycle based on the first set of intensity values; determine an inter-cluster-interference metric estimating light interference from the first cluster of oligonucleotides on the second cluster of oligonucleotides based on the set of illumination indicators; and generate, for the sequencing cycle, a modified second set of intensity values for the second signal from the second cluster of oligonucleotides by removing the inter-cluster- interference metric from the second set of intensity values.

2. The system of claim 1, further comprising instructions that, when executed by the at least one processor, cause the system to: determine the set of illumination indicators based further on amplitudes for the first set of intensity values and an estimated point spread function for a section of a nucleotide-sample slide comprising the first cluster of oligonucleotides; and determine the inter-cluster-interference metric based further on the estimated point spread function.

3. The system of claim 2, wherein the estimated point spread function uses a location of the second cluster of oligonucleotides or a different cluster of oligonucleotides as a point and includes an area comprising the first cluster of oligonucleotides and one or more other clusters of oligonucleotides.

4. The system of claim 1, wherein a first location within a nucleotide-sample slide for the first cluster of oligonucleotides is first adjacent, second adjacent, or third adjacent to a second location within the nucleotide-sample slide for the second cluster of oligonucleotides.

5. The system of claim 1, further comprising instructions that, when executed by the at least one processor, cause the system to:determine, for the sequencing cycle, a nucleobase call for the first cluster of oligonucleotides based on the first set of intensity values and intensity -value boundaries for nucleobases; and determine the set of illumination indicators based further on the nucleobase call for the first cluster of oligonucleotides.

6. The system of claim 5, further comprising instructions that, when executed by the at least one processor, cause the system to: determine the nucleobase call for the first cluster of oligonucleotides based on an intensity value from the first set of intensity values corresponding to a first channel and an intensity value from the first set of intensity values corresponding to a second channel; and generate the modified second set of intensity values by subtracting values for the inter- cluster-interference metric from an intensity value from the second set of intensity values corresponding to the first channel or an intensity value from the second set of intensity values corresponding to the second channel.

7. The system of claim 1, further comprising instructions that, when executed by the at least one processor, cause the system to determine the set of illumination indicators by: determining a first illumination indicator indicating whether the first cluster of oligonucleotides is illuminated or not illuminated in a first channel during the sequencing cycle; and determining a second illumination indicator indicating whether the second cluster of oligonucleotides is illuminated or not illuminated in a second channel during the sequencing cycle; or determining a first continuous illumination indicator indicating a degree to which the first cluster of oligonucleotides is illuminated in the first channel during the sequencing cycle; and determining a second continuous illumination indicator indicating a degree to which the first cluster of oligonucleotides is illuminated in the second channel during the sequencing cycle.

8. The system of claim 1, further comprising instructions that, when executed by the at least one processor, cause the system to determine, for the sequencing cycle and based on the modified second set of intensity values, an adjusted set of illumination indicators that represents whether the second cluster of oligonucleotides is illuminated during the sequencing cycle and that differs from an initial set of illumination indicators corresponding to the second set of intensity values.

9. The system of claim 1, further comprising instructions that, when executed by the at least one processor, cause the system to determine, for the sequencing cycle and based on themodified second set of intensity values, a nucleobase call for the second cluster of oligonucleotides that differs from a nucleobase corresponding to the second set of intensity values.

10. The system of claim 1, further comprising instructions that, when executed by the at least one processor, cause the system to: detect, for the sequencing cycle, a third set of intensity values for a third signal from a third cluster of oligonucleotides; determine an additional set of illumination indicators representing whether the third cluster of oligonucleotides is illuminated during the sequencing cycle based on the third set of intensity values; determine an additional inter-cluster-interference metric estimating light interference from the third cluster of oligonucleotides on the second cluster of oligonucleotides based on the additional set of illumination indicators; and generate, for the sequencing cycle, the modified second set of intensity values for the second signal from the second cluster of oligonucleotides by removing the inter-cluster- interference metric and the additional inter-cluster-interference metric from the second set of intensity values.

11. The system of claim 1, further comprising instructions that, when executed by the at least one processor, cause the system to: determine the first set of intensity values for the first signal from the first cluster of oligonucleotides is within an intensity-value range; determine the second set of intensity values for the second signal from the second cluster of oligonucleotides is not within the intensity-value range; and based on the first set of intensity values being within the intensity -value range and the second set of intensity values not being within the intensity-value range, generate the modified second set of intensity values by removing, from the second set of intensity values, the inter-cluster- interference metric estimating light interference from the first cluster of oligonucleotides on the second cluster of oligonucleotides.

12. The system of claim 11, further comprising instructions that, when executed by the at least one processor, cause the system to: determine, based on the first set of intensity values, a first nucleobase call for the first cluster of oligonucleotides as part of a first subset of oligonucleotide clusters having intensity values within the intensity-value range; and determine, based on the modified second set of intensity values, a second nucleobase call for the second cluster of oligonucleotides as part of a second subset of oligonucleotide clusters having intensity values not within the intensity-value range.

13. The system of claim 1, further comprising instructions that, when executed by the at least one processor, cause the system to: detect the first set of intensity values by detecting, in a single channel, a first intensity value for the first signal from a first cluster of oligonucleotides; detect the second set of intensity values by detecting, in the single channel, a second intensity value for the second signal from a second cluster of oligonucleotides; and determine the set of illumination indicators by determining a single illumination indicator representing whether the first cluster of oligonucleotides is illuminated in the single channel during the sequencing cycle.

14. A non-transitory computer-readable medium storing instructions thereon that, when executed by at least one processor, cause a computing device to: detect, for a sequencing cycle, a first set of intensity values for a first signal from a first cluster of oligonucleotides and a second set of intensity values for a second signal from a second cluster of oligonucleotides; determine a set of illumination indicators representing whether the first cluster of oligonucleotides is illuminated during the sequencing cycle based on the first set of intensity values; determine an inter-cluster-interference metric estimating light interference from the first cluster of oligonucleotides on the second cluster of oligonucleotides based on the set of illumination indicators; and generate, for the sequencing cycle, a modified second set of intensity values for the second signal from the second cluster of oligonucleotides by removing the inter-cluster-interference metric from the second set of intensity values.

15. The non-transitory computer-readable medium of claim 14, further comprising instructions that, when executed by the at least one processor, cause the computing device to: determine the set of illumination indicators based further on amplitudes for the first set of intensity values and an estimated point spread function for a section of a nucleotide-sample slide comprising the first cluster of oligonucleotides; and determine the inter-cluster-interference metric based further on the estimated point spread function.

16. The non-transitory computer-readable medium of claim 15, wherein the estimated point spread function uses a location of the second cluster of oligonucleotides or a different cluster of oligonucleotides as a point and includes an area comprising the first cluster of oligonucleotides and one or more other clusters of oligonucleotides.

17. The non-transitory computer-readable medium of claim 14, wherein a first location within a nucleotide-sample slide for the first cluster of oligonucleotides is first adjacent, secondadjacent, or third adjacent to a second location within the nucleotide-sample slide for the second cluster of oligonucleotides.

18. The non-transitory computer-readable medium of claim 14, further comprising instructions that, when executed by the at least one processor, cause the computing device to: determine, for the sequencing cycle, a nucleobase call for the first cluster of oligonucleotides based on the first set of intensity values and intensity -value boundaries for nucleobases; and determine the set of illumination indicators based further on the nucleobase call for the first cluster of oligonucleotides.

19. The non-transitory computer-readable medium of claim 18, further comprising instructions that, when executed by the at least one processor, cause the computing device to: determine the nucleobase call for the first cluster of oligonucleotides based on an intensity value from the first set of intensity values corresponding to a first channel and an intensity value from the first set of intensity values corresponding to a second channel; and generate the modified second set of intensity values by subtracting values for the inter- cluster-interference metric from an intensity value from the second set of intensity values corresponding to the first channel or an intensity value from the second set of intensity values corresponding to the second channel.

20. The non-transitory computer-readable medium of claim 14, further comprising instructions that, when executed by the at least one processor, cause the computing device to determine the set of illumination indicators by: determining a first illumination indicator indicating whether the first cluster of oligonucleotides is illuminated or not illuminated in a first channel during the sequencing cycle; and determining a second illumination indicator indicating whether the second cluster of oligonucleotides is illuminated or not illuminated in a second channel during the sequencing cycle; or determining a first continuous illumination indicator indicating a degree to which the first cluster of oligonucleotides is illuminated in the first channel during the sequencing cycle; and determining a second continuous illumination indicator indicating a degree to which the first cluster of oligonucleotides is illuminated in the second channel during the sequencing cycle.

21. The non-transitory computer-readable medium of claim 14, further comprising instructions that, when executed by the at least one processor, cause the computing device to determine, for the sequencing cycle and based on the modified second set of intensity values, an adjusted set of illumination indicators that represents whether the second cluster of oligonucleotidesis illuminated during the sequencing cycle and that differs from an initial set of illumination indicators corresponding to the second set of intensity values.

22. The non-transitory computer-readable medium of claim 14, further comprising instructions that, when executed by the at least one processor, cause the computing device to determine, for the sequencing cycle and based on the modified second set of intensity values, a nucleobase call for the second cluster of oligonucleotides that differs from a nucleobase corresponding to the second set of intensity values.

23. The non-transitory computer-readable medium of claim 14, further comprising instructions that, when executed by the at least one processor, cause the computing device to: detect, for the sequencing cycle, a third set of intensity values for a third signal from a third cluster of oligonucleotides; determine an additional set of illumination indicators representing whether the third cluster of oligonucleotides is illuminated during the sequencing cycle based on the third set of intensity values; determine an additional inter-cluster-interference metric estimating light interference from the third cluster of oligonucleotides on the second cluster of oligonucleotides based on the additional set of illumination indicators; and generate, for the sequencing cycle, the modified second set of intensity values for the second signal from the second cluster of oligonucleotides by removing the inter-cluster- interference metric and the additional inter-cluster-interference metric from the second set of intensity values.

24. The non-transitory computer-readable medium of claim 14, further comprising instructions that, when executed by the at least one processor, cause the computing device to: determine the first set of intensity values for the first signal from the first cluster of oligonucleotides is within an intensity-value range; determine the second set of intensity values for the second signal from the second cluster of oligonucleotides is not within the intensity-value range; and based on the first set of intensity values being within the intensity -value range and the second set of intensity values not being within the intensity-value range, generate the modified second set of intensity values by removing, from the second set of intensity values, the inter-cluster- interference metric estimating light interference from the first cluster of oligonucleotides on the second cluster of oligonucleotides.

25. The non-transitory computer-readable medium of claim 24, further comprising instructions that, when executed by the at least one processor, cause the computing device to:determine, based on the first set of intensity values, a first nucleobase call for the first cluster of oligonucleotides as part of a first subset of oligonucleotide clusters having intensity values within the intensity-value range; and determine, based on the modified second set of intensity values, a second nucleobase call for the second cluster of oligonucleotides as part of a second subset of oligonucleotide clusters having intensity values not within the intensity-value range.

26. The non-transitory computer-readable medium of claim 14, further comprising instructions that, when executed by the at least one processor, cause the computing device to: detect the first set of intensity values by detecting, in a single channel, a first intensity value for the first signal from a first cluster of oligonucleotides; detect the second set of intensity values by detecting, in the single channel, a second intensity value for the second signal from a second cluster of oligonucleotides; and determine the set of illumination indicators by determining a single illumination indicator representing whether the first cluster of oligonucleotides is illuminated in the single channel during the sequencing cycle.

27. A method comprising: detecting, for a sequencing cycle, a first set of intensity values for a first signal from a first cluster of oligonucleotides and a second set of intensity values for a second signal from a second cluster of oligonucleotides; determining a set of illumination indicators representing whether the first cluster of oligonucleotides is illuminated during the sequencing cycle based on the first set of intensity values; determining an inter-cluster-interference metric estimating light interference from the first cluster of oligonucleotides on the second cluster of oligonucleotides based on the set of illumination indicators; and generating, for the sequencing cycle, a modified second set of intensity values for the second signal from the second cluster of oligonucleotides by removing the inter-cluster- interference metric from the second set of intensity values.

28. The method of claim 27, further comprising: determining the set of illumination indicators based further on amplitudes for the first set of intensity values and an estimated point spread function for a section of a nucleotide-sample slide comprising the first cluster of oligonucleotides; and determining the inter-cluster-interference metric based further on the estimated point spread function.

29. The method of claim 28, wherein the estimated point spread function uses a location of the second cluster of oligonucleotides or a different cluster of oligonucleotides as a point andincludes an area comprising the first cluster of oligonucleotides and one or more other clusters of oligonucleotides.

30. The method of claim 27, wherein a first location within a nucleotide-sample slide for the first cluster of oligonucleotides is first adjacent, second adjacent, or third adjacent to a second location within the nucleotide-sample slide for the second cluster of oligonucleotides.

31. The method of claim 27, further comprising: determining, for the sequencing cycle, a nucleobase call for the first cluster of oligonucleotides based on the first set of intensity values and intensity -value boundaries for nucleobases; and determining the set of illumination indicators based further on the nucleobase call for the first cluster of oligonucleotides.

32. The method of claim 31, further comprising: determining the nucleobase call for the first cluster of oligonucleotides based on an intensity value from the first set of intensity values corresponding to a first channel and an intensity value from the first set of intensity values corresponding to a second channel; and generating the modified second set of intensity values by subtracting values for the inter- cluster-interference metric from an intensity value from the second set of intensity values corresponding to the first channel or an intensity value from the second set of intensity values corresponding to the second channel.

33. The method of claim 27, wherein determining the set of illumination indicators comprises: determining a first illumination indicator indicating whether the first cluster of oligonucleotides is illuminated or not illuminated in a first channel during the sequencing cycle; and determining a second illumination indicator indicating whether the second cluster of oligonucleotides is illuminated or not illuminated in a second channel during the sequencing cycle; or determining a first continuous illumination indicator indicating a degree to which the first cluster of oligonucleotides is illuminated in the first channel during the sequencing cycle; and determining a second continuous illumination indicator indicating a degree to which the first cluster of oligonucleotides is illuminated in the second channel during the sequencing cycle.

34. The method of claim 27, further comprising determining, for the sequencing cycle and based on the modified second set of intensity values, an adjusted set of illumination indicators that represents whether the second cluster of oligonucleotides is illuminated during the sequencingcycle and that differs from an initial set of illumination indicators corresponding to the second set of intensity values.

35. The method of claim 27, further comprising determining, for the sequencing cycle and based on the modified second set of intensity values, a nucleobase call for the second cluster of oligonucleotides that differs from a nucleobase corresponding to the second set of intensity values.

36. The method of claim 27, further comprising: detecting, for the sequencing cycle, a third set of intensity values for a third signal from a third cluster of oligonucleotides; determining an additional set of illumination indicators representing whether the third cluster of oligonucleotides is illuminated during the sequencing cycle based on the third set of intensity values; determining an additional inter-cluster-interference metric estimating light interference from the third cluster of oligonucleotides on the second cluster of oligonucleotides based on the additional set of illumination indicators; and generating, for the sequencing cycle, the modified second set of intensity values for the second signal from the second cluster of oligonucleotides by removing the inter-cluster- interference metric and the additional inter-cluster-interference metric from the second set of intensity values.

37. The method of claim 27, further comprising: determining the first set of intensity values for the first signal from the first cluster of oligonucleotides is within an intensity-value range; determining the second set of intensity values for the second signal from the second cluster of oligonucleotides is not within the intensity-value range; and based on the first set of intensity values being within the intensity -value range and the second set of intensity values not being within the intensity-value range, generating the modified second set of intensity values by removing, from the second set of intensity values, the inter-cluster- interference metric estimating light interference from the first cluster of oligonucleotides on the second cluster of oligonucleotides.

38. The method of claim 37, further comprising: determining, based on the first set of intensity values, a first nucleobase call for the first cluster of oligonucleotides as part of a first subset of oligonucleotide clusters having intensity values within the intensity-value range; anddetermining, based on the modified second set of intensity values, a second nucleobase call for the second cluster of oligonucleotides as part of a second subset of oligonucleotide clusters having intensity values not within the intensity-value range.

39. The method of claim 27, wherein: detecting the first set of intensity values comprises detecting, in a single channel, a first intensity value for the first signal from a first cluster of oligonucleotides; detecting the second set of intensity values comprises detecting, in the single channel, a second intensity value for the second signal from a second cluster of oligonucleotides; and determining the set of illumination indicators comprises determining a single illumination indicator representing whether the first cluster of oligonucleotides is illuminated in the single channel during the sequencing cycle.

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