Methods for genome assembly, haplotype phasing, and target independent nucleic acid detection

• US 10,457,934 B2
• Filed: 07/13/2017
• Issued: 10/29/2019
• Est. Priority Date: 10/19/2015
• Status: Active Grant

• Current Assignee: Dovetail Genomics, Llc
• Original Assignee: Dovetail Genomics, Llc
• Inventors: Green, Richard E., Hartley, Paul, Troll, Christopher, Min, Ei Ei
• Primary Examiner(s): Woitach, Joseph
• Application Number:
  US15/649,268
• Publication Number:
  US 20170314014A1
• Time in Patent Office: 838 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• 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 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• 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 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• 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 50/00 ICT programming tools or database systems specially adapted for bioinformatics
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

The disclosure provides methods to assemble genomes of eukaryotic or prokaryotic organisms. The disclosure provides methods for haplotype phasing and meta-genomics assemblies. The disclosure provides a streamlined method for accomplishing these tasks, such that intermediates need not be labeled by an affinity label to facilitate binding to a solid surface. The disclosure also provides methods and compositions for the de novo generation of scaffold information, linkage information, and genome information for unknown organisms in heterogeneous metagenomic samples or samples obtained from multiple individuals. Practice of the methods can allow de novo sequencing of entire genomes of uncultured or unidentified organisms in heterogeneous samples, or the determination of linkage information for nucleic acid molecules in samples comprising nucleic acids obtained from multiple individuals.

Claims

1. A method of assaying for nucleic acid species diversity in a heterogeneous sample comprising at least two species, comprising

a) obtaining a stabilized nucleic acid sample comprising a diverse plurality of nucleic acids from at least two species stabilized such that, for at least a first member of the plurality, a first nucleic acid segment and a second nucleic acid segment are held together independent of their common phosphodiester backbone, wherein said phosphodiester backbone is cleaved between said first nucleic acid segment and said second nucleic acid segment, and for at least a second member of the plurality, a third nucleic acid segment and a fourth nucleic acid segment are held together independent of their common phosphodiester backbone, wherein said phosphodiester backbone is cleaved between said third nucleic acid segment and said fourth nucleic acid segment;

b) tagging said first nucleic acid segment with a first tag and said second nucleic acid segment with a second tag, such that said first nucleic acid segment and said second nucleic acid segment are identifiable as arising from a common nucleic acid of the diverse plurality of nucleic acids, and tagging said third nucleic acid segment with a third tag and said fourth nucleic acid segment with a fourth tag, such that said third nucleic acid segment and said fourth nucleic acid segment are identifiable as arising from a common nucleic acid of the diverse plurality of nucleic acids;

c) sequencing at least an identifiable portion of said first nucleic acid segment and said first tag, of said second nucleic acid segment and said second tag, of said third nucleic acid segment and said third tag, and of said fourth nucleic acid segment and said fourth tag;

d) constructing at least a first sequence scaffold comprising said first nucleic acid segment and said second nucleic acid segment and a second sequence scaffold comprising said third nucleic acid segment and said fourth nucleic acid segment;

such that a plurality of segments of said diverse plurality of nucleic acids are assigned to at least one of the first or second sequence scaffold; and

e) counting a plurality of sequence scaffolds constructed,

wherein nucleic acid segments tagged such that they are identifiable as arising from a common nucleic acid of the diverse plurality of nucleic acids are assigned to a common scaffold; and

wherein the number of scaffolds generated indicates the species diversity in the heterogeneous sample.

2. The method of claim 1, wherein tagging said first nucleic acid segment and said second nucleic acid segment comprises adding a first oligo to the first nucleic acid segment and adding a second oligo to the second segment, said first oligo and said second oligo sharing a common oligo sequence.

3. The method of claim 2, wherein nucleic acid segments having said common oligo sequence are assigned to a common scaffold.

4. The method of claim 3, comprising mapping said identifiable portion of said first nucleic acid segment to a contig dataset, and including any matching contig of said contig dataset into said common scaffold.

5. The method of claim 4, wherein the contig dataset is concurrently generated.

6. The method of claim 4, wherein the contig dataset is obtained from a database.

7. The method of claim 1, wherein tagging said first nucleic acid segment and said second nucleic acid segment comprises ligating said first nucleic acid segment to said second nucleic acid segment, and wherein said first nucleic acid segment and said second nucleic acid segment are assigned to a common scaffold.

8. The method of claim 7, comprising mapping said identifiable portion of said first nucleic acid segment to a contig dataset, and including any matching contig of said contig dataset into said common scaffold.

9. The method of claim 8, wherein the contig dataset is concurrently generated.

10. The method of claim 8, wherein the contig dataset is obtained from a database.

11. The method of claim 1, wherein said phosphodiester backbone is cleaved subsequent to said obtaining said stabilized nucleic acid sample.

12. The method of claim 1, wherein said stabilized nucleic acid sample is contacted to a crosslinking agent.

13. The method of claim 1, wherein said stabilized nucleic acid sample is an FFPE sample.

14. The method of claim 1, comprising contacting said heterogeneous sample to a reverse transcriptase.

15. The method of claim 1, comprising categorizing said common scaffold as corresponding to a sample condition when a plurality of samples correlating to said condition have said common scaffold and if a plurality of samples lacking said condition lack said sample.

16. The method of claim 1, wherein sequence reads from the nucleic acid segments assemble into at least two nucleic acid scaffolds without reference to exogenous sequence information.

17. The method of claim 1, wherein sequence reads from the nucleic acid segments assemble into at least two nucleic acid scaffolds, such that at least 50% of a first genome and at least 50% of a second genome are represented in said at least two nucleic acid scaffolds.

18. The method of claim 1, wherein the method comprises using SPRI beads.

19. The method of claim 1, wherein the stabilized nucleic acid sample comprises no greater than about 5 micrograms of DNA.

20. The method of claim 1, wherein said first tag and said second tag are the same.

21. The method of claim 1, wherein said third tag and said fourth tag are the same.

22. The method of claim 1, wherein said first tag comprises a covalently-linked DNA segment from said second nucleic acid segment and said second tag comprises a covalently-linked DNA segment from said first nucleic acid segment.

23. The method of claim 1, wherein said third tag comprises a covalently-linked DNA segment from said fourth nucleic acid segment and said fourth tag comprises a covalently-linked DNA segment from said third nucleic acid segment.