Digital Counting of Individual Molecules by Stochastic Attachment of Diverse Label-Tags
First Claim
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.
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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.
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Citations
69 Claims
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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:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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12. The method of claim wherein 11 wherein the target is RNA and the extending step is by reverse transcriptase.
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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.
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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:
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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. - View Dependent Claims (36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47)
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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:
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(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). - View Dependent Claims (49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 66, 67, 68, 69)
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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.
Specification