JPWO2020167712A5 - - Google Patents

Description

The foregoing examples are illustrative of the invention and are not to be construed as limitations thereof. Although the invention has been described in detail with reference to preferred embodiments, variations and modifications are within the scope and spirit of the invention as described and defined in the following claims.
The claims as filed are as follows.
[Claim 1]
A non-transposase-encoding, synthetic transposon comprising a DNA barcode region that joins on its 5′ and 3′ ends to flanking regions recognized by the transposase.
[Claim 2]
2. The synthetic transposon of claim 1, which does not encode a transposase, consisting of a DNA barcode region joined on its 5' and 3' ends to flanking regions recognized by the transposase.
[Claim 3]
3. The synthetic transposon of claim 1 or 2, wherein said flanking regions comprise terminal inverted repeats.
[Claim 4]
4. The synthetic transposon of any one of claims 1-3, wherein said flanking regions comprise a DNA barcode.
[Claim 5]
5. The synthetic transposon of any one of claims 1-4, wherein the DNA barcode has a length of less than 400, 300, 200 or 50 nucleotides.
[Claim 6]
A transposome comprising the synthetic transposon of any one of claims 1 to 5 and a transposase that binds to each of the terminal inverted repeats.
[Claim 7]
7. The transposome of Claim 6, wherein said transposase is modified from a wild-type transposase.
[Claim 8]
8. The transposome of claim 7, wherein said transposase is a hyperactive mutated transposase.
[Claim 9]
9. The transposome of any one of claims 6-8, wherein the transposase is Tn5 or modified Tn5.
[Claim 10]
Two or more of the synthetic transposons according to any one of claims 1 to 5 and/or the transposons of any one of claims 6 to 9, each synthetic transposon comprising a unique DNA barcode. A library containing two or more of the posomes.
[Claim 11]
11. A kit comprising a synthetic transposon according to any one of claims 1-5, a transposome according to any one of claims 6-9, or a library according to claim 10.
[Claim 12]
12. The kit of claim 11, further comprising a transposase that recognizes the sequence of said synthetic transposon.
[Claim 13]
a) targeting an enzyme to a specific feature in chromatin in a sample;
b) activating said enzyme to alter or label DNA localized to said feature;
c) preparing said chromatin for sequencing;
d) sequencing said chromatin using long-read sequencing;
e) mapping the location of said chromatin feature based on the location of altered or labeled DNA.
[Claim 14]
14. The method of claim 13, wherein the sample comprises chromatin from less than 1000, 500, 100, 10 or 5 cells.
[Claim 15]
14. The method of claim 13, wherein said sample comprises chromatin from a single cell.
[Claim 16]
16. The method of claim 15, wherein the method is performed using a single cell droplet method.
[Claim 17]
17. The method of any one of claims 13-16, wherein the sample comprises cells.
[Claim 18]
17. The method of any one of claims 13-16, wherein the sample comprises nuclei.
[Claim 19]
19. The method of claim 17 or 18, wherein the cells or nuclei are attached to a solid support.
[Claim 20]
20. The method of claim 19, wherein said solid support is a bead or well.
[Claim 21]
21. The method of claim 20, wherein said beads are magnetic beads.
[Claim 22]
22. The method of any one of claims 17-21, further comprising permeabilizing the cells or nuclei.
[Claim 23]
17. The method of any one of claims 13-16, wherein the sample comprises chromatin isolated from cells.
[Claim 24]
24. The method of any one of claims 13-23, wherein said chromatin is obtained from a biological sample.
[Claim 25]
The biological sample is blood, serum, plasma, urine, saliva, semen, prostatic fluid, nipple aspirate, tears, sweat, feces, buccal swab, cerebrospinal fluid, cell lysate, amniotic fluid, gastrointestinal fluid, raw 25. The method of claim 24, which is a biopsy tissue, lymph or cerebrospinal fluid.
[Claim 26]
24. The method of any one of claims 13-23, wherein said chromatin is derived from a diseased tissue or sample.
[Claim 27]
24. The method of any one of claims 13-23, wherein said chromatin is derived from a peripheral tissue or cell.
[Claim 28]
28. The method of claim 27, wherein said peripheral tissue or cells are peripheral blood mononuclear cells.
[Claim 29]
29. The method of any one of claims 13-28, which maps chromatin accessibility.
[Claim 30]
30. The method of any one of claims 13-29, wherein the enzyme is a transposase.
[Claim 31]
A sample containing chromatin, the synthetic transposon according to any one of claims 1 to 5, the transposome according to any one of claims 6 to 9, or the library according to claim 10, and the 31. The method of claim 30, comprising contacting said chromatin with a synthetic transposon under conditions that allow it to insert.
[Claim 32]
32. The method of claim 31, wherein said transposase recognizes terminal inverted repeats of synthetic transposons.
[Claim 33]
33. The method of claim 32, wherein said transposase is modified from a wild-type transposase.
[Claim 34]
34. The method of claim 33, wherein said transposase is a hyperactive mutant transposase.
[Claim 35]
35. The method of any one of claims 30-34, wherein the transposase is Tn5 or modified Tn5.
[Claim 36]
36. The method of any one of claims 30-35, further comprising repairing transposon ligation sites prior to sequencing.
[Claim 37]
37. The method of any one of claims 30-36, wherein two or more samples are contacted with a synthetic transposon, each sample being contacted with a different synthetic transposon containing a unique barcode.
[Claim 38]
38. The method of claim 37, wherein two or more samples are stored after step b).
[Claim 39]
30. The method of any one of claims 13-29, wherein the enzyme is integrase or DNA methyltransferase.
[Claim 40]
29. The method of any one of claims 13-28, which maps chromatin modifications, chromatin-associated proteins, chromatin accessibility or nucleosome location.
[Claim 41]
41. The method of claim 40, wherein said chromatin modifications are histone modifications, histone variants or DNA modifications.
[Claim 42]
said histone modification is N-acetylation of serine or alanine; phosphorylation of serine, threonine or tyrosine; N-crotonylation; N-acylation of lysine; N6-methylation, N6,N6-dimethylation or N6 of lysine omega-N-methylation, symmetric or asymmetric dimethylation of arginine; citrullination of arginine; ubiquitinylation of lysine; sumoylation of lysine; O-methylation of serine or threonine. ADP-ribosylation of arginine, aspartate or glutamate, or any combination thereof.
[Claim 43]
The histone variant is H3.3, H2A. Bbd, H2A. Z. 1, H2A. Z. 2, H2A. 42. The method of claim 41 which is X, mH2A1.1, mH2A1.2, mH2A2, TH2B or any combination thereof.
[Claim 44]
42. The method of claim 41, wherein said DNA modification is 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-carboxycytosine, 3-methylcytosine, or any combination thereof.
[Claim 45]
41. The method of claim 40, wherein said chromatin associated protein is a transcription factor, histone binding protein or DNA binding protein.
[Claim 46]
46. The method of any one of claims 40-45, wherein the enzyme binds to an antibody binding protein.
[Claim 47]
47. The method of claim 46, wherein said antibody binding protein is Protein A, Protein G, a fusion between Protein A and Protein G, Protein L or Protein Y.
[Claim 48]
48. The method of any one of claims 40-47, wherein the enzyme is a transposase.
[Claim 49]
A sample containing chromatin, the synthetic transposon according to any one of claims 1 to 5, the transposome according to any one of claims 6 to 9, or the library according to claim 10, and the 49. The method of claim 48, comprising contacting said chromatin with a synthetic transposon under conditions that allow it to insert.
[Claim 50]
50. The method of claim 49, wherein said transposase recognizes terminal inverted repeats of synthetic transposons.
[Claim 51]
51. The method of claim 50, wherein said transposase is modified from a wild-type transposase.
[Claim 52]
52. The method of claim 51, wherein said transposase is a hyperactive mutant transposase.
[Claim 53]
53. The method of any one of claims 48-52, wherein the transposase is Tn5 or modified Tn5.
[Claim 54]
54. The method of any one of claims 48-53, further comprising repairing transposon ligation sites prior to sequencing.
[Claim 55]
55. The method of any one of claims 48-54, wherein two or more samples are contacted with a synthetic transposon, each sample being contacted with a different synthetic transposon containing a unique barcode.
[Claim 56]
56. The method of claim 55, wherein the two or more samples are stored after step b).
[Claim 57]
48. The method of any one of claims 40-47, wherein the enzyme is integrase or DNA methyltransferase.
[Claim 58]
Step c) is performed on the cell population, dividing the cell population into groups and sequencing using adapters containing the second barcode or PCR using primers containing the second barcode and treating the cells such that each cell contains a dual barcode signature for amplification.
[Claim 59]
59. The method of claim 58, wherein each population of cells comprises about 10 to about 30 cells.
[Claim 60]
60. The method of claim 59, wherein each cell population comprises about 20 cells.
[Claim 61]
61. The method of any one of claims 13-60, further comprising analyzing DNA modifications in chromatin using multi-omics processes.
[Claim 62]
62. The method of claim 61, wherein said DNA modification is methylation.
[Claim 63]
63. The method of any one of claims 13-62, wherein said long-read sequencing comprises nanopore sequencing.
[Claim 64]
63. The method of any one of claims 13-62, wherein said long-read sequencing comprises single-molecule real-time sequencing.
[Claim 65]
65. The method of any one of claims 13-64, further comprising mechanically or enzymatically shearing the sample prior to sequencing.
[Claim 66]
66. The method of any one of claims 13-65, further comprising amplifying the sample prior to sequencing.
[Claim 67]
66. The method of any one of claims 13-65, wherein the sample is not amplified prior to sequencing.
[Claim 68]
68. The method of any one of claims 13-67, further comprising using sequencing results to compare chromatin features between healthy and diseased tissue.
[Claim 69]
68. The method of any one of claims 13-67, further comprising using sequencing results to predict a disease state.
[Claim 70]
68. The method of any one of claims 13-67, further comprising using sequencing results to monitor response to therapy.
[Claim 71]
68. The method of any one of claims 13-67, further comprising analyzing tumor heterogeneity using sequencing results.

Claims (20)

a) targeting an enzyme to a specific feature in chromatin in a sample;
b) activating said enzyme to alter or label DNA localized to said feature;
c) preparing said chromatin for sequencing;
d) sequencing said chromatin using long-read sequencing;
e) mapping the location of said chromatin feature based on the location of altered or labeled DNA. 2. The method of claim 1 , wherein said sample comprises cells or nuclei . 3. The method of claim 2 , wherein said cells or nuclei are attached to a solid support. 4. The method of claim 3 , wherein said solid support is a magnetic bead. 5. The method of any one of claims 2-4 , further comprising the step of permeabilizing said cells or nuclei. 6. The method of any one of claims 1-5 , which maps chromatin modifications, chromatin-associated proteins, chromatin accessibility or nucleosome location. 7. The method of claim 6 , wherein said chromatin modifications are histone modifications, histone variants or DNA modifications. 7. The method of claim 6 , wherein said chromatin associated protein is a transcription factor, histone binding protein or DNA binding protein. 9. The method of any one of claims 6-8 , wherein the enzyme binds to an antibody binding protein. 10. The method of claim 9 , wherein said antibody binding protein is Protein A, Protein G, a fusion between Protein A and Protein G, Protein L or Protein Y. 11. The method of any one of claims 6-10 , wherein the enzyme is integrase or DNA methyltransferase. Step c) is performed on the cell population, dividing the cell population into groups and sequencing using adapters containing the second barcode or PCR using primers containing the second barcode and treating the cells such that each cell contains a dual barcode signature for amplification. 13. The method of any one of claims 1-12 , further comprising analyzing DNA modifications in chromatin using multi-omics processes. 14. The method of claim 13 , wherein said DNA modification is methylation. 15. The method of any one of claims 1-14 , wherein the long-read sequencing comprises nanopore sequencing. A non-transposase-encoding, synthetic transposon comprising DNA barcode regions joined on its 5′ and 3′ ends to flanking regions recognized by a transposase, said flanking regions comprising terminal inverted repeats. including, synthetic transposons. 17. A transposome comprising the synthetic transposon of claim 16 and a transposase that binds to each of the inverted terminal repeats. 18. The transposome of claim 17 , wherein said transposase is a hyperactive mutated transposase. 19. A library comprising two or more of the synthetic transposons of claim 16 and/or two or more of the transposomes of claim 17 or 18 , each synthetic transposon comprising a unique DNA barcode. 20. A kit comprising a synthetic transposon according to claim 16 , a transposome according to claim 17 or 18 , or a library according to claim 19 .