Computer-aided probability base calling for arrays of nucleic acid probes on chips

• US 6,066,454 A
• Filed: 10/10/1997
• Issued: 05/23/2000
• Est. Priority Date: 09/14/1995
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Lipshutz, Robert J., Walker, Michael G.
• Primary Examiner(s): Zitomer, Stephanie
• Assistant Examiner(s): Forman, Betty J
• Application Number:
  US08/948,896
• Time in Patent Office: 956 Days
• Field of Search: 435/6, 435/973, 536/23.1, 536/24.3, 536/24.31, 536/24.32, 935/78, 436/497, 382/129
• US Class Current: 506/8
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte

Abstract

A computer system for analyzing nucleic acid sequences is provided. The computer system is used to calculate probabilities for determining unknown bases by analyzing the fluorescence intensities of hybridized nucleic acid probes on biological chips. Additionally, information from multiple experiments is utilized to improve the accuracy of calling unknown bases.

Claims

1. In a computer system, a method of calling an unknown base in a sample nucleic acid sequence, the method comprising the steps of:

inputting a plurality of hybridization probe intensities, each of the probe intensities indicating hybridization affinity between a sample nucleic acid sequence comprising an unknown base and a nucleic acid probe;

for each probe intensity of the plurality of probe intensities, determining a probability that the nucleic acid probe for the probe intensity best hybridizes with the sample nucleic acid sequence; and

calling the unknown base according to the nucleic acid probe with the highest probability.

2. The method of claim 1, wherein the highest probability indicates a confidence that the unknown base is called correctly.

3. The method of claim 1, wherein each nucleic acid probe has a different base at an interrogation position.

4. The method of claim 3, wherein each probability indicates the likelihood that the unknown base is complementary to a base at the interrogation position in the nucleic acid probe.

5. The method of claim 1, wherein each probe intensity of the plurality of probe intensities is calculated from a plurality of intensity values.

6. The method of claim 5, wherein each probe intensity of the plurality of probe intensities is a mean of the plurality of intensity values.

7. The method of claim 5, further comprising the step of calculating a standard deviation for each intensity value of the plurality of intensity values of a probe intensity.

8. The method of claim 1, further comprising the step of comparing the highest probability to a probability threshold.

9. The method of claim 8, wherein the unknown base is called according to the nucleic acid probe with the highest probability if the highest probability exceeds the probability threshold.

10. The method of claim 8, further comprising the steps of:

producing a sum of the highest probability and a next highest probability;

comparing the sum to the probability threshold; and

calling the unknown base according to the nucleic acid probes with the highest and next highest probabilities if the sum exceeds the probability threshold.

11. The method of claim 10, wherein the sum indicates a confidence that the unknown base is called correctly if the sum exceeds the probability threshold.

12. The method of claim 1, wherein each probability is determined by a first equation:

space="preserve" listing-type="equation">Prob(X)=Prob(I.sub.x >

max(I.sub.y)).sub.Y+X

where X and Y include A, C, G, or T(U);

I is a probe intensity associated with a subscripted base; and

max represents a maximum of the probe intensities.

13. The method of claim 12, wherein the first equation is solved by a second equation:

space="preserve" listing-type="equation">prob(I.sub.X >

max(I.sub.Y))≈

Π

prob(I.sub.X >

I.sub.Y).sub.Y+X

where X and Y include A, C, G, or T(U) and Π

represents a product of probabilities that I_X is greater than other of the probabilities.

14. The method of claim 13, wherein the second equation is solved by a third equation:

##EQU4## where X and Y include A, C, G, or T(U) and a represents a standard deviation of (σ

² being a variance) of intensities for a subscripted base.

15. The method of claim 14, wherein the third equation is solved by a fourth equation:

##EQU5## where Φ

represents a density equation of standard normal distribution.

16. The method of claim 1, wherein the unknown base is called as being A, C, G, or T(U).

17. A computer program that calls an unknown base in a sample nucleic acid sequence, comprising:

code that receives as input a plurality of hybridization probe intensities, each of the probe intensities indicating hybridization affinity between a sample nucleic acid sequence comprising an unknown base and a nucleic acid probe;

code that determines for each probe intensity of the plurality of probe intensities a probability that the corresponding nucleic acid probe best hybridizes with the sample nucleic acid sequence; and

code that calls the unknown base according to the nucleic acid probe with the highest associated probability;

wherein the codes are stored on a tangible medium.

18. The computer program of claim 17, wherein the highest probability indicates a confidence that the unknown base is called correctly.

19. The computer program of claim 17, wherein each nucleic acid probe has a different base at an interrogation position.

20. The computer program of claim 19, wherein each probability indicates the likelihood that the unknown base is complementary to a base at the interrogation position in the nucleic acid probe.

21. The computer program of claim 17, wherein each probe intensity of the plurality of probe intensities is calculated from a plurality of intensity values.

22. The computer program of claim 21, wherein each probe intensity of the plurality of probe intensities is a mean of the plurality of intensity values.

23. The computer program of claim 21, further comprising the step of calculating a standard deviation for each intensity value of the plurality of intensity values of a probe intensity.

24. The computer program of claim 17, further comprising the step of comparing the highest probability to a probability threshold.

25. The computer program of claim 24, wherein the unknown base is called according to the nucleic acid probe with the highest probability if the highest probability exceeds the probability threshold.

26. The computer program of claim 24, further comprising the steps of:

producing a sum of the highest probability and a next highest probability;

comparing the sum to the probability threshold; and

calling the unknown base according to the nucleic acid probes with the highest and next highest probabilities if the sum exceeds the probability threshold.

27. The computer program of claim 26, wherein the sum indicates a confidence that the unknown base is called correctly if the sum exceeds the probability threshold.

28. A system that calls an unknown base in a sample nucleic acid sequence, comprising:

a processor; and

a computer readable medium coupled to the processor, said computer readable medium storing a computer program comprising;

code that receives as input a plurality of hybridization probe intensities, each of the probe intensities indicating hybridization affinity between a sample nucleic acid sequence comprising an unknown base and a nucleic acid probe;

code that determines for each probe intensity of the plurality of probe intensities a probability that the corresponding nucleic acid probe best hybridizes with the sample nucleic acid sequence; and

code that calls the unknown base according to the nucleic acid probe with the highest associated probability.

29. The computer program of claim 28, wherein the highest probability indicates a confidence that the unknown base is called correctly.

30. The computer program of claim 28, wherein each nucleic acid probe has a different base at an interrogation position.

31. The computer program of claim 30, wherein each probability indicates the likelihood that the unknown base is complementary to a base at the interrogation position in the nucleic acid probe.

32. The computer program of claim 28, wherein each probe intensity of the plurality of probe intensities is calculated from a plurality of intensity values.

33. The computer program of claim 32, wherein each probe intensity of the plurality of probe intensities is a mean of the plurality of intensity values.

34. The computer program of claim 32, further comprising the step of calculating a standard deviation for each intensity value of the plurality of intensity values of a probe intensity.

35. The computer program of claim 28, further comprising the step of comparing the highest probability to a probability threshold.

36. The computer program of claim 35, wherein the unknown base is called according to the nucleic acid probe with the highest probability if the highest probability exceeds the probability threshold.

37. The computer program of claim 35, further comprising the steps of:

producing a sum of the highest probability and a next highest probability;

comparing the sum to the probability threshold; and

calling the unknown base according to the nucleic acid probes with the highest and next highest probabilities if the sum exceeds the probability threshold.

38. The computer program of claim 37, wherein the sum indicates a confidence that the unknown base is called correctly if the sum exceeds the probability threshold.