METHODS FOR GENOME ASSEMBLY AND HAPLOTYPE PHASING

• US 20150363550A1
• Filed: 01/31/2014
• Published: 12/17/2015
• 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, Richard E. Jr., Lareau, Liana F.
• Granted Patent: US 10,089,437 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search
• CPC Class Codes: G16B 30/20 Sequence assembly
• CPC Class Codes: G16B 35/00 ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
• CPC Class Codes: G16B 35/10 Design of libraries
• CPC Class Codes: G16C 20/60 In silico combinatorial chemistry

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 for genome assembly comprising:

generating a plurality of contigs;

generating a plurality of read pairs from data produced by probing the physical layout of chromosomes, chromatin, or reconstituted chromatin;

mapping or assembling the plurality of read pairs to the plurality of contigs;

constructing an adjacency matrix of contigs using the read-mapping or assembly data; and

analyzing the adjacency matrix to determine a path through the contigs that represents their order and/or orientation to the genome.

2. The method of claim 1, wherein the plurality of contigs is generated by using a shotgun sequencing method, comprising:

fragmenting up long stretches of a subject'"'"'s DNA into random fragments of indeterminate size;

sequencing the fragments using high throughput sequencing methods to generate a plurality of sequencing reads; and

assembling the sequencing reads so as to form a plurality of contigs.

3. The method of claim 1, wherein the plurality of read pairs is generated by probing the physical layout of chromosomes, chromatin, or reconstituted chromatin using a Hi-C based technique.

4. The method of claim 3, wherein the Hi-C based technique comprises:

crosslinking chromosomes, chromatin, or reconstituted chromatin with a fixative agent to form DNA-protein cross links;

cutting the cross-linked DNA-Protein with one or more restriction enzymes so as to generate a plurality of DNA-protein complexes comprising sticky ends;

filling in the sticky ends with nucleotides containing one or more markers to create blunt ends that are then ligated together;

fragmenting the plurality of DNA-protein complexes into fragments;

pulling down junction containing fragments by using the one or more of markers; and

sequencing the junction containing fragments using high throughput sequencing methods to generate a plurality of read pairs.

5. The method of claim 1, wherein the plurality of read pairs is generated by probing the physical layout of chromosomes or chromatin isolated from cultured cells or primary tissue.

6. The method of claim 1, wherein the plurality of read pairs is generated by probing the physical layout of reconstituted chromatin formed by complexing naked DNA obtained from a sample of one or more subjects with isolated histones.

12. A method for determining haplotype phasing, comprising the method of claim 1, wherein the method further comprises:

identifying one or more sites of heterozygosity in the plurality of read pairs; and

identifying read pairs that comprise a pair of heterozygous sites, wherein phasing data for allelic variants can be determined from the identification of the pair of heterozygous sites.

13. A method for a meta-genomics assembly, comprising the method of claim 1, wherein the plurality of read pairs is determined by probing the physical layout of a plurality of microbial chromosomes using a modified Hi-C based method comprising:

collecting microbes from an environment; and

adding a fixative agent so as to form cross-links within each microbial cell, wherein read pairs mapping to different contigs indicate which contigs are from the same species.

14. The method of claim 13, wherein the fixative agent is formaldehyde.

7-11. -11. (canceled)

15. A method of assembling a plurality of contigs generated from a single DNA molecule, comprising:

generating a plurality of read-pairs from the single DNA molecule; and

assembling the contigs using the read-pairs, wherein at least 1 to at least 10% of the read-pairs span a distance of at least 30 kB on the single DNA molecule, and wherein the read-pairs are generated within 7 to 14 days.

16-21. -21. (canceled)

22. A method of haplotype phasing, comprising:

generating a plurality of read-pairs from a single DNA molecule; and

assembling a plurality of contigs of the DNA molecule using the read-pairs, wherein at least 1 to at least 10% of the read-pairs spans a distance of at least 30 kB on the single DNA molecule, and wherein the haplotype phasing is performed at greater than 70% accuracy.

29. The method of claim 22, wherein the haplotype phasing is performed at greater than 90% accuracy.

31. The method of claim 22, wherein the generating of a plurality of read-pair from a DNA molecule, comprises:

(a) crosslinking the first DNA molecule in vitro, wherein the DNA molecule comprises a first DNA segment and a second DNA segment;

(b) linking the first DNA segment with the second DNA segment and thereby forming a linked DNA segment; and

(c) sequencing the linked DNA segment and thereby obtaining a the first read-pair.

32. The method of claim 31, wherein a plurality of association molecules are cross-linked to the DNA molecule.

33. The method of claim 32, wherein the association molecules comprise amino acids.

34. The method of claim 33, wherein the association molecules are peptides or proteins.

35. The method of claim 31, wherein the DNA molecule is cross-linked with a fixative agent.

36. The method of claim 35, wherein the fixative agent is formaldehyde.

37. The method of claim 31, wherein the first DNA segment and the second DNA segment are generated by severing the first DNA molecule.

38. The method of claim 31, further comprising assembling a plurality of contigs of the DNA molecule using the first read-pair.

23-28. -28. (canceled)

30. (canceled)

39-74. -74. (canceled)