Methods and systems for identifying disease-induced mutations

• US 10,053,736 B2
• Filed: 10/17/2014
• Issued: 08/21/2018
• Est. Priority Date: 10/18/2013
• Status: Active Grant

• Current Assignee: Seven Bridges Genomics, Inc.
• Original Assignee: Seven Bridges Genomics, Inc.
• Inventors: Kural, Deniz
• Primary Examiner(s): Zeman, Mary K
• Application Number:
  US14/517,451
• Publication Number:
  US 20150197815A1
• Time in Patent Office: 1,404 Days
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

Abstract

The invention includes methods and systems for identifying diseased-induced mutations by producing multi-dimensional reference sequence constructs that account for variations between individuals, different diseases, and different stages of those diseases. Once constructed, these reference sequence constructs can be used to align sequence reads corresponding to genetic samples from patients suspected of having a disease, or who have had the disease and are in suspected remission. The reference sequence constructs also provide insight to the genetic progression of the disease.

Claims

1. A method of identifying cancer-induced genetic mutations, comprising using a processor coupled to a non-transitory computer-readable medium to perform:

obtaining a first nucleic acid sequence corresponding to a nucleic acid in a non-cancerous sample from a subject;

identifying differences between the first nucleic acid sequence and a selected reference sequence;

representing, in the non-transitory computer-readable storage medium, the identified differences between the first nucleic acid sequence and the selected reference sequence as two or more alternative paths in a first reference directed acyclic graph (DAG) comprising nodes, wherein each alternative path is placed at a position in the first reference DAG where there is a difference between the first nucleic acid sequence and the reference sequence;

aligning one or more sequence reads from a second sequence corresponding to a cancerous sample from the subject to the first reference DAG, wherein the aligning considers two or more alternative paths by looking backward to any prior nodes on the first reference DAG to find a maximum score for the one or more sequence reads; and

identifying, based on the aligned one or more sequence reads to the first reference DAG, differences between the second sequence and the first reference DAG as new mutations correlated with the cancer.

2. The method of claim 1, further comprising:

preparing a second reference DAG representing the differences between the first sequence, the second sequence, and the reference sequence as two or more alternative paths at positions in the second reference DAG where there is a difference between the first sequence and the reference sequence or where there is a difference between the second sequence and the first sequence;

aligning one or more reads from a third sequence corresponding to an advanced cancerous sample from the organism to the second reference DAG; and

identifying differences between the third sequence and the second reference DAG as mutations due to the advanced cancer.

3. The method of claim 1, wherein the second sequence represents a major genetic clone due to the cancer.

4. The method of claim 2, wherein the third sequence represents a minor genetic clone due to the cancer.

5. The method of claim 1, wherein the cancer is selected from breast, lung, skin, prostate, thyroid, pancreatic, bladder, or ovarian cancer.

6. The method of claim 1, wherein the cancer is leukemia or lymphoma.

7. The method of claim 1, wherein the sequence reads are at least about 50 bp in length.

8. The method of claim 1, wherein the differences between the second sequence and the first reference DAG comprise insertions, deletions, polymorphisms or structural variants.

9. The method of claim 1, wherein the reference DAG represents a genome.

10. The method of claim 1, further comprising collecting the non-cancerous non-diseased sample from the subject.

11. The method of claim 10, wherein the first nucleic acid sequence is obtained by sequencing a nucleic acid isolated from the non-cancerous sample.

12. The method of claim 1, further comprising collecting the cancerous sample from the subject.

13. The method of claim 12, wherein the one or more sequence reads from the second sequence are obtained by sequencing a nucleic acid isolated from the cancerous sample.

18. The method of claim 1, further comprising representing the new mutations correlated with the cancer as alternative paths in the reference DAG.

19. The method of claim 18, further comprising aligning one or more sequence reads from a third sequence corresponding to a sample later collected from the same subject to the first reference DAG, and monitoring cancer progression or recurrence based on the alignment.

20. The method of claim 1, further comprising reporting the new mutations correlated with the cancer to a health care provider.

14. A method of identifying mutations due to an advanced stage of cancer in a subject, comprising:

obtaining a first sequence corresponding to a non-cancerous sample from the subject and a second sequence corresponding to a cancerous sample from the subject organism;

identifying differences between the first sequence and the second sequence;

representing the identified differences between the first sequence and the second sequence as two or more alternative paths in a reference directed acyclic graph (DAG) comprising nodes, wherein each alternative path is placed at a position in the reference DAG where there is a difference between the first sequence and the second sequence;

aligning a sequence read corresponding to an advanced cancerous sample from the subject to the reference DAG, wherein the aligning considers the two or more alternative paths by looking backward to any prior nodes on the reference DAG to find a maximum score for the sequence read; and

identifying, based on the aligned sequence read to the reference DAG, differences between the sequence read and the reference DAG as new mutations correlated with an advanced stage of the cancer.

15. The method of claim 14, further comprising diagnosing the subject as having an advanced stage of the cancer.

16. The method of claim 14, wherein the cancer is selected from breast, lung, skin, prostate, thyroid, pancreatic, bladder, or ovarian cancer.

17. The method of claim 14, wherein the advanced stage of the cancer is metastatic cancer.