Analysis methods

• US 9,228,233 B2
• Filed: 09/14/2012
• Issued: 01/05/2016
• Est. Priority Date: 10/17/2011
• Status: Active Grant

• Current Assignee: Invitae Corporation
• Original Assignee: Good Start Genetics, Inc. (Invitae Corporation)
• Inventors: Kennedy, Caleb J., Umbarger, Mark, Porreca, Gregory
• Primary Examiner(s): Woolwine, Samuel
• Assistant Examiner(s): Zhang, Kaijiang
• Application Number:
  US13/616,788
• Publication Number:
  US 20130288901A1
• Time in Patent Office: 1,208 Days
• Field of Search: 435/6.1, 435/6.11, 435/6.12, 506/2, 506/7
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2563/185 Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals

Abstract

The invention generally relates to methods for analyzing nucleic acids to identify novel mutations associated with diseases. In certain embodiments, methods of the invention involve obtaining nucleic acid from a subject having a disease, identifying at least one mutation in the nucleic acid, and comparing the mutation to a database of mutations known to be associated with the disease, wherein mutations that do not match to the database are identified as novel mutations.

Claims

1. A method for identifying a novel mutation associated with a disease, the method comprising:

obtaining nucleic acid from a subject having a disease;

sequencing the nucleic acid to obtain a sequence of the nucleic acid; and

using a computer system comprising a processor coupled to a memory for;

comparing the sequence of the nucleic acid from the subject to a reference sequence, thereby determining the presence of at least one spatially ambiguous mutation in the nucleic acid;

comparing the spatially ambiguous mutation to a database of mutations known to be associated with the disease, wherein mutations that do not match to the database are identified as novel mutations;

calculating an equivalent insertion/deletion region for the spatially ambiguous mutation;

annotating the spatially ambiguous mutation with functional information;

retrieving an extreme lower position and an extreme upper position of the equivalent insertion/deletion region for the spatially ambiguous mutation; and

choosing one of the extreme lower position and the extreme upper position of the equivalent insertion/deletion region for the spatially ambiguous mutation that is outside of a functional region, wherein said spatially ambiguous mutation is not causative of the disease.

2. The method according to claim 1, wherein sequencing is sequencing-by-synthesis.

3. The method according to claim 2, wherein sequencing-by-synthesis is single molecule sequencing-by-synthesis.

4. The method according to claim 1, wherein the reference sequence is a consensus human sequence or a sequence from a non-diseased sample.

5. The method according to claim 1, wherein prior to determining the presence of the at least one spatially ambiguous mutation in the nucleic acid, the method further comprises attaching a barcode sequence to the nucleic acid.

6. The method according to claim 1, wherein the disease is cystic fibrosis.

7. The method according to claim 6, wherein the subject is Hispanic.

8. A method for identifying a novel mutation associated with a disease, the method comprising:

obtaining nucleic acid from a subject having a disease;

sequencing the nucleic acid;

comparing, using a computer system comprising a processor coupled to a memory, the sequence of the nucleic acid from the subject to a reference sequence, thereby determining the presence of a spatially ambiguous mutation in the nucleic acid; and

comparing, using the computer system, the mutation to a database of mutations known to be associated with the disease, wherein mutations that do not match to the database are identified as novel mutations;

identifying, using the computer system, a contiguous block of DNA in the reference sequence representing a tandem repeat associated with the spatially ambiguous mutation;

annotating, using the computer system, the spatially ambiguous mutation with functional information;

retrieving, using the computer system, an extreme lower position and an extreme upper position of the contiguous block of DNA in the reference sequence for the spatially ambiguous mutation; and

choosing, using the computer system, one of the lower position and the upper position of the equivalent insertion/deletion region for the spatially ambiguous mutation that is outside of a functional region, wherein said spatially ambiguous mutation is not causative of the disease.

9. The method according to claim 8, wherein sequencing is sequencing-by-synthesis.

10. The method according to claim 9, wherein sequencing-by-synthesis is single molecule sequencing-by-synthesis.

11. The method according to claim 8, wherein the reference sequence is a consensus human sequence or a sequence from a non-diseased sample.

12. The method according to claim 8, wherein prior to sequencing, the method further comprises attaching a barcode sequence to the nucleic acid.

13. The method according to claim 8, wherein the disease is cystic fibrosis.

14. The method according to claim 8, wherein the subject is Hispanic.

15. A method for determining if a mutation is causative of a disease, the method comprising:

conducting an assay to obtain a nucleic acid sequence from a subject having a disease; and

using a computer system comprising a memory coupled to a processor for;

determining the presence of a spatially ambiguous novel mutation in the sequence;

calculating an equivalent insertion/deletion region for the spatially ambiguous mutation;

annotating the mutation with appropriate functional information;

retrieving an extreme lower position and an extreme upper position of the equivalent insertion/deletion region for the mutation; and

choosing one of the extreme lower position and the extreme upper position for the spatially ambiguous mutation that is outside of a functional region, wherein said spatially ambiguous mutation is not causative of the disease.