MASSIVELY PARALLEL CONTIGUITY MAPPING

• US 20130203605A1
• Filed: 02/02/2012
• Published: 08/08/2013
• Est. Priority Date: 02/02/2011
• Status: Active Grant

• Current Assignee: University of Washington
• Original Assignee: University of Washington
• Inventors: Shendure, Jay Ashok, Schwartz, Jerrod Joseph, Adey, Andrew Colin, Lee, Cho li, Hiatt, Joseph Brian, Kitzman, Jacob Otto, Kumar, Akash
• Granted Patent: US 10,457,936 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: Y02P 20/582 Recycling of unreacted starting or intermediate materials

Abstract

Contiguity information is important to achieving high-quality de novo assembly of mammalian genomes and the haplotype-resolved resequencing of human genomes. The methods described herein pursue cost-effective, massively parallel capture of contiguity information at different scales.

Claims

1-23. -23. (canceled)

24. A method for capturing contiguity information comprising:

treating a target DNA sequence with a transposase resulting in one or more fragmentation or insertion events;

adding or inserting one or more recognition sequences to the target DNA sequence;

sequencing the treated DNA; and

capturing contiguity information by identifying target DNA sequences or recognition sequences having a shared property.

25. The method of claim 24, wherein the one or more recognition sequences are added or inserted during the transposase treatment.

26. The method of claim 24, wherein the one or more recognition sequences are added or inserted during a subsequent amplification.

27. The method of claim 24, wherein the one or more fragmentation or insertion events results in generation of a library of target nucleic acid molecules derived from the target DNA.

28. The method of claim 27, wherein the one or more recognition sequences are one or more barcodes added to the end of each target nucleic acid molecule that flank the fragmentation or insertion events.

29. The method of claim 28, wherein the shared property of the one or more barcodes is an identical or complementary barcode sequence.

30. The method of claim 24, wherein the target DNA sequence comprises a set of target DNA fragments.

31. The method of claim 30, further comprising compartmentalizing the target DNA fragments with emulsions or dilutions, generating two or more compartments of target DNA fragments prior to or after treating with the transposase.

32. The method of claim 31, wherein the one or more recognition sequences are one or more compartment-specific barcodes, each of which corresponds to the one or more compartments generated in the compartmentalizing step.

33. The method of claim 32, wherein the shared property of the one or more primer sequences is an identical compartment-specific barcode.

34. The method of claim 32, further comprising performing nucleic acid amplification using primers bearing compartment-specific barcodes.

35. The method of claim 24, wherein the one or more recognition sequences is one or more adaptor sequences that modify the ends of the target DNA sequence, or insert within the target DNA sequence.

36. The method of claim 35, wherein the one or more adaptor sequences are complementary to one or more surface-bound primers.

37. The method of claim 36, wherein the transposase is bound to a nucleic acid that is complementary to a second surface-bound primer.

38. The method of claim 36, further comprising hybridizing the one or more adaptor sequences to the one or more surface bound primers.

39. The method of claim 38, wherein the shared property is a constrained physical location.

40. The method of claim 24, wherein the transposase is bound to a surface-bound recognition sequence to form a surface-bound transposase complex.

41. The method of claim 40, wherein the shared property is a constrained physical location.

42. The method of claim 40, wherein treating the target DNA sequence comprises exposing a plurality of surface-bound transposase complexes to the target DNA sequence.

43. A method of bisulfite sequencing comprising:

a) performing in vitro transposition into target DNA molecules with transposase complexes, each transposase complex comprising a double stranded DNA transposase recognition sequence and a single stranded DNA adaptor overhang having methylated cytosine (C) residues

b) subjecting transposed target DNA molecules to bisulfite treatment;

c) performing nucleic acid amplification

d) sequencing the resulting nucleic acid library

44. The method of claim 43, further comprising incorporating a second adaptor to nucleic acid fragments derived from target DNA, after step (a) and before step (b), wherein the second adaptor is designed to facilitate nucleic acid amplification in step (c).

45. The method of claim 43, further comprising incorporating a second adaptor to nucleic acid fragments derived from target DNA, after step (b) and before step (c), wherein the second adaptor is designed to facilitate nucleic acid amplification in step (c).

46. A method for inferring chromosome conformation comprising:

a) cross-linking DNA within cells;

b) isolating cross-linked DNA from cells;

c) fragmenting the cross-linked DNA;

d) end-modifying fragmented, cross-linked DNA molecules with an adaptor that is complementary to or that corresponds to a first surface-bound primer;

e) hybridizing ends of the fragmented, end-modified target DNA molecules to the first surface-bound primer;

f) performing transposition with non-surface-bound transposase complexes, each non-surface-bound transposase complex comprising a DNA transposase and one or more sequences corresponding to a second surface-bound primer;

g) performing cluster amplification to produce clusters of clonally derived nucleic acids;

h) sequencing clusters of clonally derived nucleic acids; and

i) determining physical interactions between chromosomal positions by paring neighboring clusters together.

47. The method of claim 46, wherein the isolated cross-linked DNA are part of cross-linked DNA-protein complexes.

48. The method of claim 47, further comprising enriching for one or more specific cross-linked DNA-protein complexes by immunoprecipitation after step (c) and before step (d).