METHODS FOR GENOME ASSEMBLY, HAPLOTYPE PHASING, AND TARGET INDEPENDENT NUCLEIC ACID DETECTION

• US 20200131505A1
• Filed: 09/05/2019
• Published: 04/30/2020
• Est. Priority Date: 10/19/2015
• Status: Active Application

• Current Assignee: Dovetail Genomics, Llc
• Original Assignee: Dovetail Genomics, Llc
• Inventors: Green, Richard E., Hartley, Paul, Troll, Christopher John, Min, Ei Ei
• Application Number:
  US16/562,312
• Publication Number:
  US 20200131505A1
• 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 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

1-22. -22. (canceled)

23. A method of determining genomic linkage information for a heterogeneous nucleic acid sample comprising:

(a) obtaining a stabilized heterogeneous nucleic acid sample, wherein the stabilized heterogeneous nucleic acid sample comprises a plurality of DNA segments each of the plurality of DNA segments held together independent of their common phosphodiester backbone, wherein the common phosphodiester backbone is cleaved to create exposed DNA ends;

(b) labeling the exposed DNA ends to form labeled exposed DNA ends;

(c) ligating the labeled exposed DNA ends to form labeled paired ends;

(d) sequencing across the labeled paired ends to generate a plurality of paired sequence reads; and

(e) assigning each half of a paired sequence read of the plurality of paired sequence reads to a common nucleic acid molecule of origin.

24. The method of claim 23, wherein the stabilized heterogeneous nucleic acid sample is from a cancer sample.

25. The method of claim 23, wherein the stabilized heterogeneous nucleic acid sample is from a metagenomic sample.

26. The method of claim 23, wherein labeling the exposed DNA ends comprises adding a plurality of oligos to the exposed DNA ends.

27. The method of claim 26, wherein DNA segments of the plurality of DNA segments having a common oligo sequence are assigned to a common scaffold.

28. The method of claim 27, comprising mapping each half of the paired sequence read to a contig dataset, and including any matching contig of the contig dataset into the common scaffold.

29. The method of claim 28, wherein the contig dataset is concurrently generated.

30. The method of claim 28, wherein the contig dataset is obtained from a database.

31. The method of claim 23, wherein labeling the exposed DNA ends comprises ligating at least a first exposed DNA end to at least a second exposed DNA end of the plurality of DNA segments.

32. The method of claim 31, comprising mapping each half of the paired sequence read to a contig dataset, and including any matching contig of the contig dataset into the common scaffold.

33. The method of claim 32, wherein the contig dataset is concurrently generated.

34. The method of claim 32, wherein the contig dataset is obtained from a database.

35. The method of claim 23, wherein the common phosphodiester backbone is cleaved subsequent to obtaining the stabilized heterogeneous nucleic acid sample.

36. The method of claim 23, wherein the stabilized heterogeneous nucleic acid sample is contacted to a crosslinking agent.

37. The method of claim 23, wherein the stabilized heterogeneous nucleic acid sample is an FFPE sample.

38. The method of claim 23, comprising contacting the stabilized heterogeneous nucleic acid sample to a reverse transcriptase.

39. The method of claim 23, wherein sequence reads from the DNA 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 the at least two nucleic acid scaffolds.

40. The method of claim 23, wherein the common nucleic acid molecule of origin maps to a single individual.

41. The method of claim 23, wherein the common nucleic acid molecule of origin identifies a subset of a population.

42. The method of claim 23, wherein the method comprises using SPRI beads.

43. The method of claim 23, wherein the stabilized heterogeneous nucleic acid sample comprises no greater than about 5 micrograms of DNA.