Computer-aided visualization and analysis system for sequence evaluation

• US 6,733,964 B1
• Filed: 03/27/1998
• Issued: 05/11/2004
• Est. Priority Date: 10/21/1994
• Status: Expired due to Fees

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Lipshutz, Robert J., Chee, Mark S., Bernhart, Derek H., Jevons, Luis C., Wang, Chunwei
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US09/049,805
• Time in Patent Office: 2,237 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/287.2, 702/20
• US Class Current: 435/6.11
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00695 Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/00711 Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G16B 25/00 : ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/20 : Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
• CPC Class Codes: G16B 30/00 : ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/20 : Sequence assembly

Abstract

A computer system for analyzing nucleic acid sequences is provided. The computer system is used to perform multiple methods for determining unknown bases by analyzing the fluorescence intensities of hybridized nucleic acid probes. The results of individual experiments are improved by processing nucleic acid sequences together. Comparative analysis of multiple experiments is also provided by displaying reference sequences in one area and sample sequences in another area on a display device.

Claims

1. A computer implemented method of identifying an unknown base in a sample nucleic acid sequence, said method comprising the steps of:

inputting a plurality of probe intensities for an array of nucleic acid probes that differ from each other by a single base at an internal interrogation position into a computer system, each probe intensity indicating an extent of hybridization of a nucleic acid probe with at least one nucleic acid sequence including said sample sequence;

said computer system comparing said plurality of probe intensities to each other, wherein said comparing step includes the step of said computer system calculating a ratio of a higher probe intensity to a lower probe intensity; and

said computer system generating a base call identifying said unknown base according to results of said comparing step and said sequences of said nucleic acid probes.

2. The method of claim 1, wherein said generating step includes the step of identifying said unknown base according to a base in a nucleic acid probe having said higher probe intensity if said ratio is greater than a predetermined ratio value.

3. The method of claim 2, wherein said predetermined ratio value is approximately 1.2.

7. The method of claim 1, further comprising the step of indicating that another unknown base is unable to be identified if said plurality of probe intensities have insufficient intensity to identify said another unknown base.

8. The method of claim 1, wherein said unknown base is identified as being A, C, G, or T.

9. The method of claim 1, wherein the plurality of probe intensities are fluorescent intensities.

43. The method of claim 1, further comprising the step of setting an indicated intensity equal to a positive number if said intensity is less than or equal to zero.

4. A computer implemented method of identifying an unknown base in a sample nucleic acid sequence, said method comprising the steps of:

inputting a plurality of probe intensities for an array of nucleic acid probes that differ from each other by a single base at an internal interrogation position into a computer system, each probe intensity indicating an extent of hybridization of a nucleic acid probe with at least one nucleic acid sequence including said sample sequence;

said computer system comparing said plurality of probe intensities to each other;

sorting said plurality of probe intensities by intensity before said comparing step; and

said computer system generating a base call identifying said unknown base according to results of said comparing step and said sequences of said nucleic acid probes.

5. The method of claim 4, wherein said at least one sequence includes a reference sequence.

6. The method of claim 5, wherein said comparing step includes the step of said computer system comparing probe intensities of a probe hybridizing with said sample sequence to said probe hybridizing with said reference sequence.

15. The method of claim 5, wherein probe intensities of probes hybridizing with said reference sequence are from a plurality of experiments.

10. A computer implemented method of identifying an unknown base in a sample nucleic acid sequence, said method comprising the steps of:

inputting a plurality of probe intensities for an array of nucleic acid probes that differ from each other by a single base at an internal interrogation position into a computer system, each probe intensity indicating an extent of hybridization of a nucleic acid probe with at least one nucleic acid sequence including said sample sequence and a reference sequence;

said computer system comparing said plurality of probe intensities to each other, wherein said comparing step includes the step of calculating first ratios of a wild-type probe intensity to each probe intensity of probes hybridizing with said reference sequence, wherein said wild-type probe intensity indicates an extent of hybridization of a complementary probe with said reference sequence; and

said computer system generating a base call identifying said unknown base according to results of said comparing step and said sequences of said nucleic acid probes.

11. The method of claim 10, wherein said comparing step includes the step of calculating second ratios of the highest probe intensity of a probe hybridizing with said sample sequence to each probe intensity of probes hybridizing with said sample sequence.

12. The method of claim 11, wherein said comparing step includes the step of calculating ratios of said first ratios to said second ratios.

13. The method of claim 12, wherein said generating step includes the step of identifying said unknown base according to a base of said probe associated with a highest of said ratios of said first ratios to said second ratios.

14. The method of claim 11, wherein said comparing step includes the step of calculating a ratio of a highest probe intensity of a probe hybridizing with said reference sequence to a highest intensity of a probe hybridizing with said sample sequence.

16. A computer implemented method of identifying an unknown base in a sample nucleic acid sequence, said method comprising the steps of:

inputting a plurality of probe intensities for an array of nucleic acid probes that differ from each other by a single base at an internal interrogation position into a computer system, each probe intensity indicating an extent of hybridization of a nucleic acid probe with at least one nucleic acid sequence including said sample sequence and a reference sequence;

said computer system comparing said plurality of probe intensities to each other, wherein probe intensities of probes hybridizing with said reference sequence are from a plurality of experiments and said comparing step includes the step of said computer system comparing probe intensities of probes hybridizing with said sample sequence to statistics about said plurality of experiments; and

said computer system generating a base call identifying said unknown base according to results of said comparing step and said sequences of said nucleic acid probes.

17. The method of claim 16, wherein said statistics include a mean and standard deviation.

18. A computer implemented method of identifying an unknown base in a sample nucleic acid sequence, said method comprising the steps of:

inputting a plurality of probe intensities for an array of nucleic acid probes that differ from each other by a single base at an internal interrogation position into a computer system, each probe intensity indicating an extent of hybridization of a nucleic acid probe with at least one nucleic acid sequence including said sample sequence and a reference sequence;

normalizing said plurality of probe intensities by dividing each probe intensity by a sum of related probe intensities, wherein related probe intensities are from all the probes that differ by a single base;

said computer system comparing said plurality of probe intensities to each other, wherein probe intensities of probes hybridizing with said reference sequence are from a plurality of experiments; and

said computer system generating a base call identifying said unknown base according to results of said comparing step and said sequences of said nucleic acid probes.

19. A computer implemented method of identifying an unknown base in a sample nucleic acid sequence, said method comprising the steps of:

inputting a plurality of probe intensities for an array of nucleic acid probes that differ from each other by a single base at an internal interrogation position into a computer system, each probe intensity indicating an extent of hybridization of a nucleic acid probe with at least one nucleic acid sequence including said sample sequence;

subtracting a background intensity from each of said plurality of probe intensities;

said computer system comparing said plurality of probe intensities to each other; and

said computer system generating a base call identifying said unknown base according to results of said comparing step and said sequences of said nucleic acid probes.

20. A computer implemented method of identifying an unknown base in a sample nucleic acid sequence, said method comprising the steps of:

inputting a plurality of probe intensities for an array of nucleic acid probes that differ from each other by a single base at an interrogation position into a computer system, each probe intensity indicating an extent of hybridization of a nucleic acid probe with said sample sequence;

said computer system calculating a ratio of a higher probe intensity to a lower probe intensity; and

said computer system generating a base call identifying said unknown base according to a base of a nucleic acid probe having said higher probe intensity if said ratio is greater than a predetermined ratio value.

21. The method of claim 20, wherein said predetermined ratio value is approximately 1.2.

22. The method of claim 20, further comprising the step of sorting said plurality of probe intensities by intensity before said comparing step.

23. The method of claim 20, further comprising the step of subtracting a background intensity from each of said plurality of probe intensities.

24. The method of claim 20, further comprising the step of setting an indicated intensity equal to a positive number if said intensity is less than or equal to zero.

25. The method of claim 20, further comprising the step of indicating that another unknown base is unable to be identified if said plurality of probe intensities have insufficient intensity to identify said another unknown base.

26. The method of claim 20, wherein said unknown base is identified as being A, C, G, or T.

27. A computer implemented method of identifying an unknown base in a sample nucleic acid sequence, said method comprising the steps of:

inputting of first set of probe intensities for an array of nucleic acid probes that differ from each other by a single base at an interrogation position into a computer system, each probe intensity in said first set indicating an extent of hybridization of a nucleic acid probe with a reference nucleic acid sequence;

inputting second set of probe intensities for an array of nucleic acid probes differ from each other by a single base at an interrogation position into said computer system, each probe intensity in said second set indicating an extent of hybridization of a nucleic acid probe with said sample sequence;

said computer system comparing at least one of said probe intensities in said first set and at least one of said probe intensities in said second set, wherein said comparing step includes the step of calculating first ratios of a wild-type probe intensity to each said probe intensity of probes hybridizing with said reference sequence, wherein said wild-type probe intensity indicates an extent of hybridization of a complementary probe with said reference sequence; and

said computer system generating a base call identifying said unknown base according to results of said comparing step.

28. The method of claim 27, wherein said comparing step includes the step of calculating second ratios of the highest probe intensity of probes hybridizing with said sample sequence to each probe intensity of a probe hybridizing with said sample sequence.

29. The method of claim 28, wherein said comparing step further includes the step of calculating ratios of said first ratios to said second ratios.

30. The method of claim 29, wherein said generating step includes the step of identifying said unknown base according to a base of said probe associated with a highest of said ratios of said first ratios to said second ratios.

33. The method of claim 27, further comprising the step of setting an indicated intensity equal to a positive number if said intensity is less than or equal to zero.

35. The method of claim 27, wherein said unknown base is identified as being A, C, G, or T.

31. A computer implemented method of identifying an unknown base in a sample nucleic acid sequence, said method comprising the steps of:

inputting of first set of probe intensities for an array of nucleic acid probes that differ from each other by a single base at an interrogation position into a computer system, each probe intensity in said first set indicating an extent of hybridization of a nucleic acid probe with a reference nucleic acid sequence;

inputting second set of probe intensities for an array of nucleic acid probes differ from each other by a single base at an interrogation position into said computer system, each probe intensity in said second set indicating an extent of hybridization of a nucleic acid probe with said sample sequence;

said computer system comparing at least one of said probe intensities in said first set and at least one of said probe intensities in said second set, wherein said comparing step includes the step of calculating a ratio of a highest probe intensity in said first set to a highest intensity in said second set; and

said computer system generating a base call identifying said unknown base according to results of said comparing step.

32. A computer implemented method of identifying an unknown base in a sample nucleic acid sequence, said method comprising the steps of:

inputting of first set of probe intensities for an array of nucleic acid probes that differ from each other by a single base at an interrogation position into a computer system, each probe intensity in said first set indicating an extent of hybridization of a nucleic acid probe with a reference nucleic acid sequence;

inputting second set of probe intensities for an array of nucleic acid probes differ from each other by a single base at an interrogation position into said computer system, each probe intensity in said second set indicating an extent of hybridization of a nucleic acid probe with said sample sequence;

subtracting a background intensity from each of said plurality of probe intensities;

said computer system comparing at least one of said probe intensities in said first set and at least one of said probe intensities in said second set; and

said computer system generating a base call identifying said unknown base according to results of said comparing step.

34. A computer implemented method of identifying an unknown base in a sample nucleic acid sequence, said method comprising the steps of:

inputting of first set of probe intensities for an array of nucleic acid probes that differ from each other by a single base at an interrogation position into a computer system, each probe intensity in said first set indicating an extent of hybridization of a nucleic acid probe with a reference nucleic acid sequence;

inputting second set of probe intensities for an array of nucleic acid probes differ from each other by a single base at an interrogation position into said computer system, each probe intensity in said second set indicating an extent of hybridization of a nucleic acid probe with said sample sequence;

said computer system comparing at least one of said probe intensities in said first set and at least one of said probe intensities in said second set;

said computer system generating a base call identifying said unknown base according to results of said comparing step; and

indicating that another unknown base is unable to be identified if said plurality of probe intensities have insufficient intensity to identify said another unknown base.

36. A computer implemented method of identifying an unknown base in a sample nucleic acid sequence, said method comprising the steps of:

inputting statistics about a plurality of experiments into a computer system, each of said experiments producing probe intensities for an array of nucleic acid probes that differ from each other by a single base at an interrogation position, each probe intensity indicating an extent of hybridization of a nucleic acid probe with a reference nucleic acid sequence;

inputting a plurality of probe intensities for an array of nucleic acid probes that differ from each other by a single base at an interrogation position, each probe intensity indicating an extent of hybridization of a nucleic acid probe with said sample sequence;

said computer system comparing at least one of said plurality of probe intensities with said statistics; and

said computer system generating a base call identifying said unknown base according to results of said comparing step.

37. The method of claim 36, wherein said statistics include a mean and standard deviation.

38. The method of claim 36, further comprising the step of normalizing said plurality of probe intensities by dividing each probe intensity by a sum of related probe intensities, wherein related probe intensities are from all the probes that differ by a single base.

39. The method of claim 36, further comprising the step of subtracting a background intensity from each of said plurality of probe intensities.

40. The method of claim 36, further comprising the step of setting an indicated intensity equal to a positive number if said intensity is less than or equal to zero.

41. The method of claim 36, further comprising the step of indicating that another unknown base is unable to be identified if said plurality of probe intensities have insufficient intensity to identify said another unknown base.

42. The method of claim 36, wherein said unknown base is identified as being A, C, G, or T.