Methods for genome assembly and haplotype phasing

• US 10,529,443 B2
• Filed: 11/27/2018
• Issued: 01/07/2020
• Est. Priority Date: 02/01/2013
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Green, Jr., Richard E., Lareau, Liana F.
• Primary Examiner(s): Thomas, David C
• Application Number:
  US16/202,042
• Publication Number:
  US 20190147978A1
• Time in Patent Office: 406 Days
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• 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

The disclosure provides methods to assemble genomes of eukaryotic or prokaryotic organisms. The disclosure further provides methods for haplotype phasing and meta-genomics assemblies.

Claims

1. A method comprising:

contacting a sample to a stabilizing agent, said sample comprising a nucleic acid molecule complexed to at least one nucleic acid binding protein;

cleaving the nucleic acid into a plurality of segments comprising at least a first segment and a second segment;

attaching the first segment and the second segment at a junction;

obtaining at least some sequence on each side of the junction to generate a first read pair;

mapping the first read pair to a set of contigs; and

determining a path through the set of contigs that represents an order and/or orientation to a genome, wherein the path through the set of contigs that represents an order and/or orientation to the genome is determined so that each contig is visited exactly once.

2. The method of claim 1, comprising:

excluding the first read pair from contig assembly analysis because said at least some sequence on each side of the junction maps to a common contig; and

determining a path through the contigs that represents an order and/or orientation to the genome using a second read pair.

3. The method of claim 1, comprising:

contacting the sample to an antibody prior to contacting the sample to the stabilizing agent.

4. The method of claim 1, comprising:

contacting the sample to an antibody subsequent to contacting the sample to the stabilizing agent.

5. The method of claim 1, wherein cleaving the nucleic acid into at least a first segment and a second segment comprises cutting the nucleic acid using at least one restriction enzyme.

6. The method of claim 1, wherein cleaving the nucleic acid into at least a first segment and a second segment comprises shearing the nucleic acid.

7. The method of claim 1, wherein cleaving the nucleic acid into at least a first segment and a second segment comprises sonicating the nucleic acid.

8. The method of claim 1, wherein contacting a sample to a stabilizing agent comprises administering ultraviolet light.

9. The method of claim 1, wherein contacting a sample to a stabilizing agent comprises contacting the sample to a chemical fixative.

10. The method of claim 9, wherein the chemical fixative comprises formaldehyde.

11. The method of claim 9, wherein the chemical fixative comprises psoralen.

12. The method of claim 1, wherein the at least one nucleic acid binding protein comprises a native chromatin constituent.

13. The method of claim 1, wherein the at least one nucleic acid binding protein comprises an exogenously supplied histone.

14. The method of claim 1, wherein attaching comprises filling in sticky ends using at least some biotin tagged nucleotides and ligating the blunt ends.

15. The method of claim 1, wherein determining a path through the set of contigs that represents an order and/or orientation to the genome comprises down-weighing contigs that represent promiscuous regions of the genome.

16. The method of claim 1, wherein the set of contigs is generated by a shotgun sequencing method.

17. The method of claim 1, wherein determining a path through the set of contigs that represents an order and/or orientation to the genome comprises haplotype phasing the set of contigs.

18. The method of claim 17, wherein haplotype phasing the set of contigs comprise identifying one or more sites of heterozygosity in a plurality of read pair sequences, wherein phasing data for allelic variants is determined by identifying read pairs that comprise a pair of heterozygous sites.

19. The method of claim 1, comprising excluding from analysis read pair sequence that maps to a common contig, so as to determine a path through the set of contigs that represents an order and/or orientation to the genome using read pairs that map to different contigs.

20. A method comprising:

contacting a sample to a stabilizing agent, said sample comprising a nucleic acid molecule complexed to at least one nucleic acid binding protein;

cleaving the nucleic acid into a plurality of segments comprising at least a first segment and a second segment;

attaching the first segment and the second segment at a junction;

obtaining at least some sequence on each side of the junction to generate a first read pair;

mapping the first read pair to a set of contigs; and

determining a path through the set of contigs that represents an order and/or orientation to a genome, wherein determining a path through the set of contigs that represents an order and/or orientation to the genome comprises down-weighing contigs that represent promiscuous regions of the genome.

21. The method of claim 20, comprising:

excluding the first read pair from contig assembly analysis because said at least some sequence on each side of the junction maps to a common contig; and

determining a path through the contigs that represents an order and/or orientation to the genome using a second read pair.

22. The method of claim 20, comprising:

contacting the sample to an antibody prior to contacting the sample to the stabilizing agent.

23. The method of claim 20, comprising:

contacting the sample to an antibody subsequent to contacting the sample to the stabilizing agent.

24. The method of claim 20, wherein cleaving the nucleic acid into at least a first segment and a second segment comprises cutting the nucleic acid using at least one restriction enzyme.

25. The method of claim 20, wherein cleaving the nucleic acid into at least a first segment and a second segment comprises shearing the nucleic acid.

26. The method of claim 20, wherein cleaving the nucleic acid into at least a first segment and a second segment comprises sonicating the nucleic acid.

27. The method of claim 20, wherein contacting a sample to a stabilizing agent comprises administering ultraviolet light.

28. The method of claim 20, wherein contacting a sample to a stabilizing agent comprises contacting the sample to a chemical fixative.

29. The method of claim 28, wherein the chemical fixative comprises formaldehyde.

30. The method of claim 28, wherein the chemical fixative comprises psoralen.

31. The method of claim 20, wherein the at least one nucleic acid binding protein comprises a native chromatin constituent.

32. The method of claim 20, wherein the at least one nucleic acid binding protein comprises an exogenously supplied histone.

33. The method of claim 20, wherein attaching comprises filling in sticky ends using at least some biotin tagged nucleotides and ligating the blunt ends.

34. The method of claim 20, wherein the path through the set of contigs that represents an order and/or orientation to the genome is determined so that each contig is visited exactly once.

35. The method of claim 20, wherein the set of contigs is generated by a shotgun sequencing method.

36. The method of claim 20, wherein determining a path through the set of contigs that represents an order and/or orientation to the genome comprises haplotype phasing the set of contigs.

37. The method of claim 36, wherein haplotype phasing the set of contigs comprise identifying one or more sites of heterozygosity in a plurality of read pair sequences, wherein phasing data for allelic variants is determined by identifying read pairs that comprise a pair of heterozygous sites.

38. The method of claim 20, comprising excluding from analysis read pair sequence that maps to a common contig, so as to determine a path through the set of contigs that represents an order and/or orientation to the genome using read pairs that map to different contigs.