Computer-aided visualization and analysis system for sequence evaluation

• US 5,974,164 A
• Filed: 10/16/1995
• Issued: 10/26/1999
• Est. Priority Date: 10/21/1994
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Chee, Mark S.
• Primary Examiner(s): Couso, Jose L.
• Assistant Examiner(s): Bella, Matthew C.
• Application Number:
  US08/531,137
• Time in Patent Office: 1,471 Days
• Field of Search: 382/129, 382/178, 382/179, 435/5, 435/6, 435/91.1, 435/91.2, 435/7.1-7.2, 435/23.1, 536/24.3-24.33, 536/23.1, 364/96-99
• US Class Current: 382/129
• 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 (1) 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 may be improved by processing nucleic acid sequences together. Comparative analysis of multiple experiments is also provided by displaying reference sequences in one area (814) and sample sequences in another area (816) on a display device (3).

Claims

1. In a computer system, a method of identifying mutations in a sample nucleic acid sequence, said method comprising the steps of:

inputting a first set of probe intensities, each of said probe intensities in said first set being associated with a nucleic acid probe and indicating hybridization affinity between said associated nucleic acid probe and a reference nucleic acid sequence;

inputting a second set of probe intensities, each of said probe intensities in said second set being associated with a nucleic acid probe and indicating hybridization affinity between said associated nucleic acid probe and said sample sequences;

said computer system comparing probe intensities in said first set and probe intensities in said second set to select hybridization regions where said probe intensities in said first set and said probe intensities in said second set differ, each region including multiple base positions; and

identifying mutations in said sample sequence according to characteristics of said selected regions.

2. The method of claim 1, wherein said selected regions are determined by comparing probe intensities of wild-type probes.

3. The method of claim 2, wherein said wild-type probes are complementary to a portion of said reference sequence.

4. The method of claim 1, wherein said identifying step includes the steps of:

analyzing a selected region;

identifying a likely position of a mutation in said selected region according to an interrogation position of a nucleic acid probe; and

performing base calling at said likely position.

5. The method of claim 1, wherein said identifying step further includes the steps of:

analyzing a width of a selected region; and

determining there is a single base mutation in said selected region if said width is equal to a probe length.

6. The method of claim 5, wherein said identifying step further includes the step of identifying a mismatch zone for a mutation in said selected region according to an interrogation position of a nucleic acid probe.

7. The method of claim 6, wherein said identifying step further includes the step of performing base calling in said mismatch zone.

8. The method of claim 6, wherein said identifying step further includes the step of ignoring mutation base calls in said selected region that are not in said mismatch zone.

9. The method of claim 1, wherein said identifying step further includes the steps of:

analyzing a width of a selected region; and

determining a base mutation is not present in said selected region if said width is less than a probe length.

10. The method of claim 1, wherein said identifying step further includes the steps of:

analyzing a width of a selected region; and

determining multiple base mutations are present in said selected region if said width is greater than a probe length.

11. The method of claim 1, wherein said identifying step further includes the steps of:

analyzing a shape of a selected region; and

identifying a base mutation in said selected region according to said shape.

12. The method of claim 11, further including the step of pattern matching said shape to known shapes.

13. A computer program for identifying mutations in a sample nucleic acid sequence, comprising:

computer readable code that receives as input a first set of probe intensities, each of said probe intensities in said first set being associated with a nucleic acid probe and indicating hybridization affinity between said associated nucleic acid probe and a reference nucleic acid sequence;

computer readable code that receives as input a second set of probe intensities, each of said probe intensities in said second set being associated with a nucleic acid probe and indicating hybridization affinity between said associated nucleic acid probe and said sample sequence;

computer readable code that compares probe intensities in said first set and probe intensities in said second set to select hybridization regions where said probe intensities in said first set and said probe intensities in said second set differ, each region including multiple base positions;

computer readable code that identifies mutations in said sample sequence according to characteristics of said selected regions; and

a tangible medium that stores said computer readable codes.

14. In a computer system, a method of identifying bases in a sample nucleic acid sequence, said method of comprising the steps of:

inputting reference hybridization intensities of a reference nucleic acid sequence hybridizing with a set of probes;

inputting sample hybridization intensities of said sample sequence hybridizing with said set of probes;

selecting a region including multiple base positions where said reference hybridization intensities differ from said sample hybridization intensities; and

identifying a base in said sample sequence that differs from said reference sequence by analyzing said region.

15. The method of claim 14, wherein said set of probes is complementary to a portion of said reference sequence.

16. The method of claim 14, wherein said region is selected as having a bubble shape.

17. The method of claim 14, wherein said identifying step includes the steps of:

analyzing said selected region;

identifying a likely position of a base in said sample sequence that differs from said reference sequence in said selected region according to an interrogation position of said set of probes; and

performing base calling at said likely position.

18. The method of claim 14, wherein said identifying step further includes the steps of:

analyzing a width of said selected region; and

determining there is a single base in said sample sequence that differs from said reference sequence in said selected region if said width is equal to a probe length of said set of probes.

19. The method of claim 18, wherein said identifying step further includes the step of identifying a mismatch zone for said single base in said selected region according to an interrogation position of said set of probes.

20. The method of claim 19, wherein said identifying step further includes the step of performing base calling in said mismatch zone.

21. The method of claim 19, wherein said identifying step further includes the step of calling bases of said sample sequence in said selected region that are not in said mismatch zone as being the same as said reference sequence.

22. The method of claim 14, wherein said identifying step further includes the steps of:

analyzing a width of said selected region; and

determining said sample sequence does not differ from said reference sequence in said selected region if said width is less than a probe length of said set of probes.

23. The method of claim 14, wherein said identifying step further includes the steps of:

analyzing a width of said selected region; and

determining multiple bases in said sample sequence differ from said reference sequence in said selected region if said width is greater than a probe length of said set of probes.

24. The method of claim 14, wherein said identifying step further includes the steps of:

analyzing a shape of said selected region; and

identifying a base in said sample sequence that differs from said reference sequence in said selected region according to said shape.

25. The method of claim 24, further including the step of pattern matching said shape to know shapes.

27. The method of claim 14, further comprising the step of comparing said reference hybridization intensities to said sample hybridization intensities in order to identify regions of said reference and sample sequences where said reference hybridization intensities and said sample hybridization intensities differ.

26. A computer program product for identifying bases in a sample nucleic acid sequence, comprising:

computer readable codes that receive as input reference hybridization intensities of a reference nucleic acid sequence hybridizing with a set of probes;

computer readable code that receives as input sample hybridization intensities of said sample sequence hybridization with said set of probes;

computer readable code that selects a region including multiple base positions where said reference hybridization intensities differ from said sample hybridization intensities;

computer readable code that identifies a base in said sample sequence that differs from said reference sequence by analyzing said region; and

a tangible medium that stores said computer readable codes.