Methods and systems for uniform enrichment of genomic regions

• US 8,383,338 B2
• Filed: 02/23/2009
• Issued: 02/26/2013
• Est. Priority Date: 04/24/2006
• Status: Active Grant

• Current Assignee: Roche NimbleGen, Inc. (Roche Holding AG)
• Original Assignee: Roche NimbleGen, Inc. (Roche Holding AG)
• Inventors: Kitzman, Jacob, Richmond, Todd, D'Ascenzo, Mark, Albert, Thomas, Rodesch, Matthew, Jeddeloh, Jeffrey, Middle, Christina
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US12/391,001
• Publication Number:
  US 20090221438A1
• Time in Patent Office: 1,464 Days
• Field of Search: 435/6, 536/23.1, 536/24.3
• US Class Current: 435/6.1
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase

Abstract

The present invention provides methods and compositions for the enrichment of target nucleic acids in a microarray system. In particular, the present invention provides methods and compositions for uniform enrichment of target nucleic acid molecules in a microarray format. The present invention also provides for intentionally non-uniform enrichment among target nucleic acid molecules.

Claims

1. A method for uniform enrichment of a population of nucleic acid molecules in a sample, comprising:

a) providing a sample of nucleic acid molecules comprising a plurality of target nucleic acid sequences,

b) hybridizing the sample to nucleic acid probes complementary to the target nucleic acid sequences under hybridization conditions, wherein at least one of the target nucleic acid sequences exhibits enrichment bias for a complementary probe, and wherein the frequency of individual nucleic acid probe sequences is increased for a target nucleic acid sequence having below-average enrichment and decreased for a target nucleic acid sequence having above-average enrichment such that the nucleic acid probes provide uniform hybridization among said plurality of target nucleic acid sequences, and

c) separating non-hybridized nucleic acid sequences from hybridized target nucleic acid sequences, thereby uniformly enriching a population of nucleic acid molecules in a sample.

2. The method of claim 1, wherein said separating comprises washing said support.

3. The method of claim 1, further comprising fragmenting said sample of nucleic acid molecules prior to said hybridizing.

4. The method of claim 3, further comprising ligating an adaptor molecule to one or both ends of a plurality of fragmented nucleic acid molecules prior to said hybridizing.

5. The method of claim 4, further comprising denaturing said sample of nucleic acid molecules prior to said hybridizing.

6. The method of claim 1, further comprising eluting a plurality of hybridized target nucleic acid sequences from the support.

7. The method of claim 6, further comprising sequencing the eluted target nucleic acid sequences.

8. The method of claim 1, wherein said nucleic acid probes are immobilized on a support.

9. The method of claim 8, wherein said support is a microarray slide or a bead.

10. The method of claim 1, wherein said population of nucleic acid molecules is a population of genomic DNA molecules.

11. The method of claim 1, wherein said population of nucleic acid molecules is a population of amplified genomic DNA molecules.

12. The method of claim 1, wherein the frequency is determined by utilizing an empirically-fit linear regression model.

13. A process for uniform enrichment of a population of nucleic acid sequences in a sample, said process comprising hybridizing a plurality of immobilized hybridization probes to a plurality of corresponding target nucleic acid sequences within a population of nucleic acid molecules and separating non-hybridized nucleic acid sequences from hybridized target nucleic acid sequences, wherein at least one of the target nucleic acid sequences exhibits enrichment bias for a complementary probe, and wherein the frequency of individual hybridization probes is increased for target nucleic acid sequences having below-average enrichment and decreased for target nucleic acid sequences having above-average enrichment such that the nucleic acid probes provide uniform hybridization among said plurality of target nucleic acid sequences.

14. The process of claim 13, further comprising eluting said hybridized target nucleic acid sequences.

15. The process of claim 13, wherein the immobilized hybridization probes are immobilized on a microarray slide or a bead.

16. The process of claim 15, wherein the frequency is determined utilizing an empirically-fit linear regression model.

17. The process of claim 13, wherein said target nucleic acid sequences are genomic DNA sequences.

18. A method for uniform enrichment of a population of nucleic acid molecules in a sample, comprising:

a) providing a sample of nucleic acid molecules comprising a plurality of target nucleic acid sequences,

b) hybridizing the sample to a support comprising immobilized nucleic acid probes under conditions to support hybridization between the immobilized nucleic acid probes and the plurality of target nucleic acid sequences, wherein said immobilized nucleic acid probes are complementary to said plurality of target nucleic acid sequences, wherein the density of said immobilized nucleic acid probes to optimally produce uniform read depth is predicted using an empirically-fit, linear regression model fitting read depth to the density of immobilized nucleic acid probes, and wherein the immobilized nucleic acid probes provide uniform hybridization among said plurality of target nucleic acid sequences, and

c) separating non-hybridized nucleic acid sequences from hybridized target nucleic acid sequences, thereby uniformly enriching a population of nucleic acid molecules in a sample.

19. The method of claim 18, wherein said separating comprises washing said support.

20. The method of claim 18, further comprising fragmenting said sample of nucleic acid molecules prior to said hybridizing.

21. The method of claim 20, further comprising ligating an adaptor molecule to one or both ends of a plurality of fragmented nucleic acid molecules prior to said hybridizing.

22. The method of claim 21, further comprising denaturing said sample of nucleic acid molecules prior to said hybridizing.

23. The method of claim 18, further comprising eluting a plurality of hybridized target nucleic acid sequences from the support.

24. The method of claim 23, further comprising sequencing the eluted target nucleic acid sequences.

25. The method of claim 18, wherein said support is selected from the group consisting of a microarray slide and a bead.

26. The method of claim 18, wherein said population of nucleic acid molecules is a population of genomic DNA molecules.

27. The method of claim 18, wherein said population of nucleic acid molecules is a population of amplified genomic DNA molecules.