Methods for genome assembly and haplotype phasing
View Patent ↗The disclosure provides methods to assemble genomes of eukaryotic or prokaryotic organisms. The disclosure further provides methods for haplotype phasing and meta-genomics assemblies.
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.