Digital Counting of Individual Molecules by Stochastic Attachment of Diverse Label-Tags

• US 20130116130A1
• Filed: 12/15/2011
• Published: 05/09/2013
• Est. Priority Date: 12/15/2009
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Fu, Glenn K., Fodor, Stephen P.A.
• Granted Patent: US 9,315,857 B2
• Time in Patent Office: Days
• US Class Current: 506/4
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• 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 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2537/16 Assays for determining copy number or wherein the copy number is of special importance
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels

Abstract

Compositions, methods and kits are disclosed for high-sensitivity counting of individual molecules by stochastic labeling of a identical molecules in mixtures of molecules by attachment of a unique label-tags from a diverse pool of label tags to confer uniqueness to otherwise identical or indistinguishable events. Individual occurrences of target molecules randomly choose from a non-depleting reservoir of diverse label-tags. Labeled molecules may be detected by hybridization or sequencing based methods. Molecules that would otherwise be identical in information content are labeled to create a separately detectable product that can be distinctly detected. The disclosed stochastic transformation methods reduce the problem of counting molecules from one of locating and identifying identical molecules to a series of binary digital questions detecting whether preprogrammed label-tags are present. The methods may be used, for example, to count a given species of molecule within a sample.

Claims

1. A method for counting individual occurrences of at least one species of target molecule in a mixture of nucleic acids having an unknown number of target molecule occurrences, the method comprising:

(a) combining the mixture with a pool of label-tags, wherein the pool of label-tags comprises multiple copies of each of a plurality of different label-tag sequences;

(b) attaching label-tags from the pool of label-tags to individual occurrences of the at least one species of target molecule in a random manner to obtain label-tag-target molecules, wherein different occurrences of a species of target molecule attach to different label-tag sequences from the plurality of different label-tag sequences, thereby obtaining a plurality of unique label-tag-target molecules;

(c) optionally amplifying the unique label-tag-target molecules;

(d) detecting each type of unique label-tag-target molecule; and

(e) counting the number of unique label-tag-target molecules that are detected to obtain a count of the target molecule occurrences of the at least one species of target molecule in the mixture.

2. The method of claim 1 wherein the collection of label-tags comprises a number of different label-tag sequences that is at least twice the number of each target molecule to be estimated.

3. The method of claim 1 wherein the collection of label-tags comprises between 900 and 10,000 different label-tag sequences

4. The method of claim 1 wherein the unique label-tag-target molecules are amplified by PCR in a multiplex amplification reaction so that there are a plurality of copies of each unique label-tag-target molecule that are detected in step (d).

5. The method of claim 1 wherein between 2 and 100 different species of target molecules are counted.

6. The method of claim 1 wherein between 100 and 10,000 different species of target molecules are counted.

7. The method of claim 1 wherein between 10,000 and 50,000 different species of target molecules are counted.

8. The method of claim 1 wherein the mixture of nucleic acids is a genomic DNA sample, the method further comprising:

fragmenting the genomic DNA sample to obtain fragments comprising target molecules prior to step (a),

ligating a first adaptor comprising a common priming sequence to a first end of the fragments;

attaching the label-tags by ligating a second adaptor comprising a label-tag and a second common priming sequence to the second end of the fragments, wherein the second adaptor is randomly selected from a pool of second adaptors that comprises the pool of label-tags, wherein the label-tag is between the target fragment and the second common priming sequence;

thereby obtaining the plurality of unique label-tag-target molecules; and

amplifying the unique label-tag-target molecules by PCR using a primer or a pair of primers to the first and second common priming sequences prior to detecting step (d).

9. The method of claim 1 wherein the number of different label-tags is at least an order of magnitude larger than the expected number of target molecules.

10. The method of claim 1 wherein label-tags are attached to targets by ligation.

11. The method of claim 1 wherein the pool of label-tags comprises a pool of label-tag primers that each consist of at least one label-tag and a priming sequence that is complementary to a species of target molecule and wherein the attaching step comprises:

(i) hybridizing individual label-tag primers to individual occurrences of the target and (ii) extending the hybridized primers using the target as template to obtain unique primer extension products each comprising a different label-tag and each corresponding to a single occurrence of the target molecule.

14. The method of claim 1 wherein the ratio of target molecule occurrences to number of different label-tags is less than 1:

10.

15. The method of claim 1 wherein the ratio of target molecule occurrences to number of different label-tags is less than 1:

100.

16. The method of claim 1 wherein the ratio of target molecule occurrences to number of different label-tags is less than 1:

1000.

17. The method of claim 1 wherein the ratio of target molecule occurrences to number of different label-tags is less than 1:

10,000.

18. The method of claim 1 wherein the step of detecting each type of unique label-tag-target molecule is by hybridizing at least a portion of the label-tag-target molecules to an array of probes comprising a plurality of features wherein each feature contains a different probe sequence and the probe sequence in each feature is specific for a different label-target combination, wherein the array comprises a plurality of features that are complementary to a predicted junction between the label-tag and a region of the target and wherein features that are hybridized to label-tag-target molecules are detectably labeled.

19. The method of claim 18 wherein each feature is a different bead.

20. The method of claim 19 wherein the step of counting comprises counting the number of beads that are detectably labeled.

21. The method of claim 19 wherein the beads are present in the array at random locations and the sequence of the probes on each bead is unknown.

22. The method of claim 1 wherein the step of detecting each type of unique label-tag-target molecule is by sequencing at least a portion of the label-tag-target molecules, wherein enough of the label-tag is sequences to unambiguously identify the label-tag and enough of the target is sequenced to unambiguously identify the target species.

23. The method of claim 22 wherein sequencing is by a method selected from sequencing by synthesis, sequencing by ligation, pyrosequencing, and sequencing by hybridization.

24. The method of claim 22 wherein sequencing is by a method that sequences single molecules as they pass through a nanopore.

25. The method of claim 1 wherein the mixture of nucleic acids is the total mRNA from a single cell.

26. The method of claim 1 wherein the mixture of nucleic acids is the nucleic acid from 10 to 1,000 cells, the nucleic acid from 10 to 1,000 cells or the RNA from a 10 to 1,000 cells.

27. The method of claim 1 wherein the step of amplifying comprises a method selected from PCR, reverse transcription, in vitro transcription, rolling circle amplification, and isothermal strand displacing amplification.

12. The method of claim wherein 11 wherein the target is RNA and the extending step is by reverse transcriptase.

13. The method of claim wherein 11 wherein the target is DNA and the extending step is by a DNA dependent polymerase.

28. An array comprising a plurality of feature sets, including a feature set for each of a plurality of different target species, wherein each feature set comprises a plurality of different features each feature comprising a plurality of oligonucleotide probes of the same sequence, and wherein the features of a feature set have a common target specific sequence and a variable label-tag sequence, wherein each different feature in the set has the same target specific sequence and a different label-tag specific sequence and wherein each feature set has a feature for each different label-tag in a set of label-tags.

29. The array of claim 28 wherein the array has a first feature set for a first target species, a second feature set for a second target species and a third feature set for a third target species and wherein each feature set comprises at least one feature for each label-tag in a pool of label-tags.

30. The array of claim 28 wherein the array has between 3 and 100 different feature sets, each feature set being specific for a single different target species and each feature set comprising at least one feature for each label-tag in a pool of label-tags.

31. The array of claim 28 wherein the array has between 100 and 10,000 different feature sets, each feature set being specific for a single different target species and each feature set comprising at least one feature for each label-tag in a pool of label-tags.

32. The array of claim 30 wherein the label-tag set has a diversity of label-tags comprising 1,000 to 5,000 different label-tags and the label-tags are between 8 and 12 bases in length.

33. The array of claim 30 wherein the label-tag set has a diversity of label-tags comprising 5,000 to 100,000 different label-tags.

34. The array of claim 30 wherein the label-tag set has a diversity of label-tags comprising 100,000 to 1,000,000 different label-tags.

35. A method for determining a value for n for a plurality of different target species, wherein n is the number of occurrences of a first species of target molecule that are present in a sample, the method comprising:

attaching to each occurrence of the first species of target molecule, a label-tag from a set of diverse label-tags, wherein the set of diverse label-tags comprises m different label-tags and label-tags are chosen at random, thereby generating for each occurrence of the first species of target molecule, a labeled target molecule that comprises a single occurrence of the first species of target molecule and a label-tag from the set of diverse label-tags, wherein at least 90% of the labeled target molecules have a label-tag that is different from the label-tags on all of the other labeled target molecules of the first species of target molecule;

detecting each labeled target molecule by determining at least a partial sequence of the labeled target molecule, wherein at least X bases of the target molecule and at least Y bases of the label-tag are determined; and

counting the number of new molecules, thereby counting the number of molecules of the first target molecule in the sample.

36. The method of claim 35 wherein the ratio of m to n is greater than 50.

37. The method of claim 35 wherein the ratio of m to n is greater than 100.

38. The method of claim 35 wherein the ratio of m to n is greater than 500.

39. The method of claim 35 wherein the ratio of m to n is greater than 1000.

40. The method of claim 35 wherein the target molecules are proteins or peptides.

41. The method of claim 35 wherein target molecules are nucleic acids and the detecting step comprises sequencing.

42. The method of claim 41 wherein sequencing comprises sequencing by extension or sequencing by ligation.

43. The method of claim 41 wherein sequencing comprises sequencing using reversible terminator chemistry.

44. The method of claim 35 wherein the sample consists of a single cell, the nucleic acid from a single cell, the RNA from a single cell or a crude cell extract from a single cell.

45. The method of claim 35 wherein the sample consists of 10 to 1,000 cells, the nucleic acid from 10 to 1,000 cells or the RNA from a 10 to 1,000 cells.

46. The method of claim 35 wherein individual diverse label-tags from the set of diverse label-tags are attached to an occurrence of the first species of target molecules by extension of a primer that comprises the diverse label-tag using template dependent primer extension by an enzyme selected from:

DNA dependent RNA polymerase, a DNA dependent DNA polymerase and an RNA dependent RNA polymerase.

47. The method of claim 46 wherein the DNA dependent RNA polymerase is selected from T7 RNA polymerase and SP6 RNA polymerase.

48. A method for counting the number of occurrences of each of a plurality of different target molecules in a sample, the method comprising the steps of:

(a) providing a plurality of conjugates each comprising a label-tag selected from a pool of label-tags, and a target molecule, wherein the label-tag is adjacent to a known target sequence in the conjugate so that a label-tag-target junction is formed and wherein more than 90% of the target molecule are attached to a label-tag, the pool of oligonucleotide label-tags comprising a plurality of different label-tag sequences and wherein the probability that any two target molecules is labeled with the same label-tag is less than a selected threshold value;

(b) amplifying the conjugates in a multiplex amplification reaction to obtain amplified conjugates comprising label-tags;

(c) detecting each different amplified conjugate by detecting different label-tags;

(d) counting the number of different lable-tags and conjugates detected in step (c); and

,

(e) determining the number of target molecules in the sample from the number of different label-tags and conjugates counted in step (d).

49. The method of claim 48 wherein:

(i) the pool of label-tags has m different label-tags;

(ii) the first target species has n occurrences; and

(iii) following the covalent attaching step there are k first targets labeled with a label-tag that is unique in the set of labeled first targets; and

wherein the step of counting comprises empirically determining k and calculating n using k=m(1−

e<sup>−

(n/m)</sup>).

50. The method of claim 49 wherein the number of different label-tags in the pool of label-tags is n and the number of occurrences of the first target is m and wherein n/m is less than 0.10.

51. The method of claim 50 wherein n/m is less than 0.01.

52. The method of claim 50 wherein n/m is less than 0.005.

53. The method of claim 50 wherein n/m is less than 0.001.

54. The method of claim 48 wherein label-tags are selected at random in the step of ligating a label-tag to individual occurrences of first and second target nucleic acid species.

55. The method of claim 48 wherein the label-tags in the pool of label-tags are DNA, the first and second target species are DNA and the ligation comprises ligation with a ligase selected from T4 DNA ligase and E. coli DNA ligase.

56. The method of claim 48 wherein detecting step is by hybridization to an array of probes that are complementary to the label-tag-target junctions.

57. The method of claim 48 wherein the step of detecting and counting comprises:

(a) hybridizing the plurality of amplified labeled first target species and the plurality of amplified labeled second target species to an array of probes comprising a first plurality of probe features each feature complementary to a different first target-label-tag junction and a second plurality of probe features each feature complementary to different second target-label-tag junctions;

(b) detecting hybridized features; and

(c) counting the hybridized features.

58. The method of claim 57 wherein the array of probes comprises a glass support having probes complementary to the junctions.

59. The method of claim 57 wherein the array of probes comprises a plurality of beads having probes complementary to the junctions.

60. The method of claim 57 wherein the array of probes comprises a plurality of microspheres.

61. The method of claim 57 wherein the array of probes comprises a plurality of encoded microparticles where the microparticles for the first target are encoded with a first code and the microparticles for the second target are encoded with a second code.

62. The method of claim 48 wherein the step of detecting and counting comprises:

sequencing amplified labeled first target species and amplified labeled second target species, wherein at least a portion of the label-tag and at least a portion of the target specific region is sequenced, wherein the number of different sequences obtained for each target is used to determine the count of each target.

63. The method of claim 62 wherein the targets to be counted are the transcriptome of a single cell or a collection of cells of a single type.

64. The method of claim 62 wherein the targets to be counted are the number of chromosomes of a single type in a genome or the number of copies of an allele of a gene in a genome.

66. The kit of claim 63 wherein the label-tags in the pool of label-tags comprise a variable region that is the same length in all label-tags in the pool and the length is between 6 and 14 bases.

67. The kit of claim 66 wherein the label-tags in the pool of label-tags further comprise a recognition site for a restriction enzyme, a common priming site or an RNA polymerase promoter sequence.

68. The kit of claim 66 wherein the label-tags in the pool of label-tags are double stranded adaptors comprising a common priming sequence that is present in all label-tags in a pool of label-tags.

69. The kit of claim 63 further comprising an oligo dT primer, one or more sequencing primers, and a reverse transcriptase.

65. A kit comprising a pool of label-tags wherein the diversity of the pool of label-tags is selected from between 100 and 1,000, between 1,000 and 10,000, between 10,000 and 100,000, between 100,000 and 1,000,000 and greater than 1,000,000 different types of label-tags.