METHODS FOR GENOME ASSEMBLY AND HAPLOTYPE PHASING

• US 20140220587A1
• Filed: 01/31/2014
• Published: 08/07/2014
• 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 9,411,930 B2
• Time in Patent Office: Days
• US Class Current: 435/6.19
• 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 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 the plurality of read pairs to the plurality of contigs;

constructing an adjacency matrix of contigs using the read-mapping 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.

7. The method of claim 1, wherein for the plurality of read pairs, each read pair is weighted by taking a function of each read'"'"'s distance to the edge of the contig so as to incorporate a higher probability of shorter contacts than longer contacts.

8. The method of claim 1, wherein the adjacency matrix is re-scaled to down-weight the high number of contacts on contigs that represent promiscuous regions of the genome.

9. The method of claim 8, wherein the promiscuous regions of the genome includes one or more conserved binding sites for one or more agents that regulate the scaffolding interactions of chromatin.

10. The method of claim 9, where the one or more agents comprises transcriptional repressor CTCF.

11. The method of claim 1, wherein the method provides for the genome assembly of a human subject, wherein the plurality of contigs is generated from the human subject'"'"'s DNA, and wherein the plurality of read pairs is generated by using the human subject'"'"'s chromosomes or chromatin, or reconstituted chromatin made from the subject'"'"'s naked DNA.

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.