Tagging nucleic acids for sequence assembly

• US 10,526,641 B2
• Filed: 07/31/2015
• Issued: 01/07/2020
• Est. Priority Date: 08/01/2014
• Status: Active Grant

• Current Assignee: Dovetail Genomics, Llc
• Original Assignee: Dovetail Genomics, Llc
• Inventors: Fields, Andrew, Hartley, Paul, Putnam, Nicholas, Rice, Brandon, Stites, Jonathan
• Primary Examiner(s): Leith, Nancy J
• Application Number:
  US15/329,414
• Publication Number:
  US 20170335369A1
• Time in Patent Office: 1,621 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

Various approaches for generating long-distance contiguity information to facilitate contig assembly and phase determination are disclosed. Nucleic acids are assembled into complexes using binding moieties such that, when the nucleic acid backbones are cleaved, the ensuing fragments remain bound. Exposed ends are tagged and ligated either to one another or to tagging moieties such as oligo labels. Ligated junctions are sequenced, and the sequence information is used to assemble contigs into common scaffolds or to assign phase information. Various approaches to tagging the exposed ends are presented.

Claims

1. A method of nucleic acid analysis comprising:

a) providing a crosslinked DNA sample comprising a first DNA molecule, wherein said first DNA molecule comprises a first sequence segment and a second sequence segment held together independent of a common phosphodiester bond of the first DNA molecule;

b) cleaving said first DNA molecule using an endonuclease such that said first segment and said second segment no longer share a common phosphodiester backbone;

c) isolating said first sequence segment and said second sequence segment in a first reaction volume;

d) attaching a label to said first sequence segment and to said second sequence segment in said first reaction volume;

e) obtaining sequence information of said label and said first sequence segment and said second sequence segment; and

f) assigning sequence of the first sequence segment and the second sequence segment to a first data contig indicated by the label.

2. The method of claim 1, further comprising using said sequence information to perform an assembly of a genome sequence of an organism of said crosslinked DNA sample.

3. The method of claim 2, wherein said assembly is a de novo assembly.

4. The method of claim 3, wherein said de novo assembly is performed in less than 14 days.

5. The method of claim 1, wherein said first reaction volume is an aqueous droplet.

6. The method of claim 1, wherein said first sequence segment and said second sequence segment are isolated in said first reaction volume using a microfluidic device.

7. The method of claim 1, wherein said first sequence segment and said second sequence segment are held together by at least one association molecule.

8. The method of claim 7, wherein said association molecules comprise peptides or proteins.

9. The method of claim 8, wherein said peptides or proteins comprise reconstituted chromatin.

10. The method of claim 8, wherein said peptides or proteins comprise endogenous chromatin.

11. The method of claim 1, wherein said label comprises one or more elements selected from the group consisting of a primer, a barcode and a restriction site.

12. The method of claim 1, wherein said label is produced in said first reaction volume prior to or concurrent with said attaching.

13. The method of claim 1, wherein said crosslinked DNA sample is crosslinked via cross-linked proteins bound to one or more nucleotide sequence segments.

14. The method of claim 1, wherein said crosslinked DNA sample is crosslinked non-specifically.

15. The method of claim 1, comprising obtaining phasing information of said crosslinked DNA sample from said first sequence segment and said second sequence segment.

16. The method of claim 15, wherein said phasing information is obtained using information from said label.

17. The method of claim 15, wherein said phasing information comprises chromosome-level phasing information.

18. The method of claim 17, wherein said chromosome-level phasing information comprises phasing information for 90% or more of single nucleotide polymorphisms (SNPs) in said crosslinked DNA sample at an accuracy of at least 90%.

19. The method of claim 18, wherein said accuracy is at least 99%.

20. The method of claim 1, wherein said crosslinked DNA sample comprises a plurality of polynucleotides from only a single individual.

21. The method of claim 1, wherein said first reaction volume comprises no sample DNA other than said first DNA molecule.

22. The method of claim 1, wherein said crosslinked DNA sample was crosslinked outside of a cell.

23. The method of claim 1, wherein said first sequence segment and said second sequence segment are held together by at least one substrate.

24. The method of claim 23, wherein the substrate comprises a bead.