Methods for genome assembly and haplotype phasing

• US 9,910,955 B2
• Filed: 05/27/2016
• Issued: 03/06/2018
• 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:
  US15/167,880
• Publication Number:
  US 20160275242A1
• Time in Patent Office: 648 Days
• Field of Search: None
• 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 naked DNA from a sample taken from a complex biological environment;

obtaining a plurality of contigs corresponding to DNA from said sample;

complexing said 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 genome assemblies, such that contigs having common read pairs are positioned in a common genome.

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

fragmenting DNA from said sample 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 plurality of read pairs is generated by probing the physical layout of reconstituted chromatin formed by complexing naked DNA with 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 genomes in sample from a complex biological environment, and wherein the plurality of read pairs is generated from reconstituted chromatin made from the sample'"'"'s naked DNA.

8. The method of claim 7, wherein the complex biological environment comprises human gut microbes.

9. The method of claim 7, wherein the complex biological environment comprises human skin microbes.

10. The method of claim 7, wherein the complex biological environment comprises waste site microbes.

11. The method of claim 7, wherein the complex biological environment comprises an ecological environment.

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

13. The method of claim 7, wherein the genome assemblies represent the contigs'"'"' order and orientation.

14. The method of claim 7, 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.