Methods and materials for assessing allelic imbalance

• US 9,574,229 B2
• Filed: 01/29/2016
• Issued: 02/21/2017
• Est. Priority Date: 06/17/2011
• Status: Active Grant

• Current Assignee: Myriad Genetics, Inc.
• Original Assignee: Myriad Genetics, Inc.
• Inventors: Gutin, Alexander, Timms, Kirsten, Lanchbury, Jerry
• Primary Examiner(s): Martinell, James
• Application Number:
  US15/010,721
• Publication Number:
  US 20160145681A1
• Time in Patent Office: 389 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• 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 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids

Abstract

Methods and systems for detecting allelic imbalance using nucleic acid sequencing are provided.

Claims

1. An in vitro method for detecting allelic imbalance at a plurality of single nucleotide polymorphism loci, comprising:

(1) extracting DNA from a formalin-fixed paraffin-embedded sample comprising at least one tumor cell obtained from a patient to produce at least one solution comprising genomic DNA of the at least one tumor cell;

(2) enriching the at least one solution in (1) for test DNA molecules each comprising at least one locus from the plurality of single nucleotide polymorphism loci, wherein the plurality of single nucleotide polymorphism loci comprises at least 1,000 single nucleotide polymorphism loci, wherein there is at least one single nucleotide polymorphism locus from the plurality of single nucleotide polymorphism loci located on average every 5Mb within each chromosome analyzed, and wherein enriching the at least one solution comprises;

(a) (i) separating the test DNA molecules from the rest of the genomic DNA in the at least one solution by contacting genomic DNA in the at least one solution with a plurality of oligonucleotide probes, wherein there is at least one probe in the plurality of oligonucleotide probes complementary to each locus in the plurality of single nucleotide polymorphism loci and (ii) amplifying the test DNA molecules separated in (i) by PCR;

or

(b) (i) amplifying the genomic DNA in the at least one solution and (ii) separating the test DNA molecules from the rest of the amplified genomic DNA in (i) by contacting the amplified genomic DNA with a plurality of oligonucleotide probes, wherein there is at least one probe in the plurality of oligonucleotide probes complementary to each locus in the plurality of single nucleotide polymorphism loci;

or

(c) directly amplifying the test DNA molecules from the at least one solution of genomic DNA; and

(3) performing a DNA sequencing reaction on the at least one enriched solution in

(2) to detect the genotype of each locus in the plurality of single nucleotide polymorphism loci;

(4) determining the lengths of a plurality of regions of allelic imbalance in the genome of the at least one tumor cell by detecting a plurality of stretches of single nucleotide polymorphism loci in which allelic imbalance is detected in (3).

2. The method of claim 1, wherein the plurality of single nucleotide polymorphism loci comprises at least 10,000 single nucleotide polymorphism loci.

3. The method of claim 1, wherein the plurality of single nucleotide polymorphism loci comprises at least 50,000 single nucleotide polymorphism loci.

4. The method of claim 1, wherein there is at least one single nucleotide polymorphism locus located on average every 100 kb within each chromosome.

5. The method of claim 1, wherein there is at least one single nucleotide polymorphism locus located on average every 50 kb within each chromosome.

6. The method of claim 1, wherein there is at least one single nucleotide polymorphism locus located on average every 10 kb within each chromosome.

7. The method of claim 1, wherein the genomic spacing of the plurality of single nucleotide polymorphism loci is less than or equal to 50%.

8. The method of claim 1, wherein the genomic spacing of the plurality of single nucleotide polymorphism loci is less than or equal to 25%.

9. The method of claim 1, wherein the genomic spacing of the plurality of single nucleotide polymorphism loci is less than or equal to 10%.

10. The method of claim 1, wherein allelic imbalance is detected in a single nucleotide polymorphism locus where the locus has loss of heterozygosity.

11. The method of claim 1, wherein allelic imbalance is detected in a single nucleotide polymorphism locus where the locus has a major copy proportion of at least 0.51.

12. The method of claim 11, wherein the major copy proportion is at least 0.6.

13. The method of claim 11, wherein the major copy proportion is at least 0.7.

14. The method of claim 11, wherein the major copy proportion is at least 0.8.

15. The method of claim 11, wherein the major copy proportion is at least 0.9.

16. The method of claim 1, wherein each stretch comprises at least three consecutive single nucleotide polymorphism loci in which allelic imbalance is detected in (4).

17. The method of claim 1, wherein each stretch comprises at least 15 consecutive single nucleotide polymorphism loci in which allelic imbalance is detected in (4).

18. The method of claim 1, wherein the plurality of single nucleotide polymorphism loci comprises at least 2,500 single nucleotide polymorphism loci.

19. The method of claim 1, wherein there is at least one single nucleotide polymorphism locus located on average every 1 Mb within each chromosome.

20. The method of claim 1, wherein the plurality of single nucleotide polymorphism loci comprises at least 5,000 single nucleotide polymorphism loci.

21. The method of claim 1, wherein there is at least one single nucleotide polymorphism locus located on average every 500 kb within each chromosome.

22. An in vitro method for detecting allelic imbalance at a plurality of single nucleotide polymorphism loci, comprising:

(1) providing a formalin-fixed paraffin-embedded sample comprising at least one tumor cell obtained from a patient;

(2) enriching the sample in (1) for test DNA molecules each comprising at least one locus from the plurality of single nucleotide polymorphism loci, wherein the plurality of single nucleotide polymorphism loci comprises at least 1,000 single nucleotide polymorphism loci and wherein there is at least one single nucleotide polymorphism locus located on average every 5 Mb within each chromosome; and

(3) performing at least one sequencing reaction on the test DNA molecules to detect the genotype of each locus in the plurality of single nucleotide polymorphism loci to determine whether each locus in the plurality of single nucleotide polymorphism loci has allelic imbalance.

23. The method of claim 22, wherein determining whether each locus in the plurality of single nucleotide polymorphism loci has allelic imbalance in (3) comprises determining whether each locus in the plurality of single nucleotide polymorphism loci is homozygous.

24. The method of claim 23, wherein the plurality of single nucleotide polymorphism loci comprises at least 5,000 single nucleotide polymorphism loci.

25. The method of claim 23, wherein the plurality of single nucleotide polymorphism loci comprises at least 50,000 single nucleotide polymorphism loci.

26. The method of claim 23, wherein there is at least one single nucleotide polymorphism locus located on average every 1 Mb within each chromosome.

27. The method of claim 23, wherein there is at least one single nucleotide polymorphism locus located on average every 500 kb within each chromosome.

28. The method of claim 23, wherein there is at least one single nucleotide polymorphism locus located on average every 100 kb within each chromosome.

29. The method of claim 23, wherein there is at least one single nucleotide polymorphism locus located on average every 50 kb within each chromosome.

30. The method of claim 23, wherein the genomic spacing of the plurality of single nucleotide polymorphism loci is less than or equal to 50%.

31. The method of claim 23, wherein the genomic spacing of the plurality of single nucleotide polymorphism loci is less than or equal to 25%.

32. The method of claim 23, wherein the genomic spacing of the plurality of single nucleotide polymorphism loci is less than or equal to 10%.

33. The method of claim 22, wherein the plurality of single nucleotide polymorphism loci comprises at least 2,500 single nucleotide polymorphism loci.

34. The method of claim 22, wherein the plurality of single nucleotide polymorphism loci comprises at least 10,000 single nucleotide polymorphism loci.

35. The method of claim 22, wherein there is at least one single nucleotide polymorphism locus located on average every 1 Mb within each chromosome.

36. The method of claim 22, wherein there is at least one single nucleotide polymorphism locus located on average every 500 kb within each chromosome.

37. The method of claim 22, wherein there is at least one single nucleotide polymorphism locus located on average every 100 kb within each chromosome.

38. The method of claim 22, wherein there is at least one single nucleotide polymorphism locus located on average every 50 kb within each chromosome.

39. The method of claim 22, wherein the genomic spacing of the plurality of single nucleotide polymorphism loci is less than or equal to 50%.

40. The method of claim 22, wherein the genomic spacing of the plurality of single nucleotide polymorphism loci is less than or equal to 25%.

41. The method of claim 22, wherein the genomic spacing of the plurality of single nucleotide polymorphism loci is less than or equal to 10%.