Methods for Genome Assembly, Haplotype Phasing, and Target Independent Nucleic Acid Detection

• US 20170314014A1
• Filed: 07/13/2017
• Published: 11/02/2017
• 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
• Granted Patent: US 10,457,934 B2
• Time in Patent Office: Days
• 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 molecular diversity in a heterogeneous sample, comprising

a) obtaining a stabilized nucleic acid sample comprising a diverse plurality of nucleic acids stabilized such that, for at least one 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;

b) tagging said first nucleic acid segment and said second nucleic acid segment 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;

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

d) assigning said first nucleic acid segment and said second nucleic acid segment to a scaffold corresponding to said tag;

such that a plurality of segments of said diverse plurality of nucleic acids are assigned to at least one scaffold; and

e) determining a number corresponding to how many scaffolds are generated;

wherein the number of scaffolds generated corresponds to the nucleic acid molecular diversity of 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 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 data set into said common scaffold.

5. The method of claim 4, wherein the contig data set 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 data set into said common scaffold.

9. The method of claim 8, wherein the contig data set 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 the heterogeneous sample comprises a plurality of allelic variants.

12. The method of claim 1, wherein said phosphodiester backbone is cleaved subsequent to said obtaining a stabilized sample.

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

14. The method of claim 1, wherein said stabilized sample is an FFPE sample.

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

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

17. The method of claim 1, wherein the heterogeneous sample comprises nucleic acids mapping to at least two individuals of a common species.

18. The method of claim 1, wherein the heterogeneous sample comprises nucleic acids mapping to at least two species.

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

20. The method of claim 1, wherein the sequence reads 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.

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

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