Methods for genome assembly and haplotype phasing

• US 9,411,930 B2
• Filed: 01/31/2014
• Issued: 08/09/2016
• 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.
• Primary Examiner(s): Thomas, David
• Application Number:
  US14/170,339
• Publication Number:
  US 20140220587A1
• Time in Patent Office: 921 Days
• Field of Search: None
• US Class Current: 1/1
• 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 of genome assembly comprising:

obtaining a plurality of contigs;

complexing naked DNA with isolated nuclear proteins to form reconstituted chromatin;

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

mapping the plurality of read pairs to the plurality of contigs thereby producing read-mapping data; and

arranging the contigs using the read-mapping data to assemble the contigs into a genome assembly, such that contigs having common read pairs are positioned to determine a path through the contigs that represents their order to the genome.

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

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

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

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

3. The method of claim 1, wherein generating a plurality of read pairs from data produced by probing the physical layout of reconstituted chromatin comprises using crosslinking.

4. The method of claim 1, wherein generating a plurality of read pairs from data produced by probing the physical layout of reconstituted chromatin comprises:

crosslinking 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 markers; and

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

5. The method of claim 1, wherein the isolated nuclear proteins comprise isolated histones.

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

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

8. 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.

9. The method of claim 1, wherein said arranging the contigs using the read pair data comprises:

constructing an adjacency matrix of contigs using the readmapping data; and

analyzing the adjacency matrix to determine a path through the contigs that represents their order in the genome.

10. The method of claim 9, comprising analyzing the adjacency matrix to determine a path through the contigs that represents their order and orientation to the genome.

11. The method of claim 9, wherein a read pair is weighted as a function of the distance from the mapped position of its first read on a first contig to the edge of that first contig and the distance from the mapped position of its second read on a second contig to the edge of that second contig.

12. The method of claim 7, wherein the plurality of contigs is generated from the human subject'"'"'s DNA.

13. The method of claim 1, wherein the genome assembly represents the contigs'"'"' order and orientation.

14. The method of claim 1, wherein a read pair is weighted as a function of the distance from the mapped position of its first read on a first contig to the edge of that first contig and the distance from the mapped position of its second read on a second contig to the edge of that second contig.

15. The method of claim 1, wherein read pairs that map to different contigs provide data about which contigs are adjacent in a correct genome assembly.