DIGITAL COUNTING OF INDIVIDUAL MOLECULES BY STOCHASTIC ATTACHMENT OF DIVERSE LABELS
First Claim
1. A method, comprising:
- detecting each of a plurality of new first molecules and a plurality of new second molecules by detecting the label sequence present on each of the new first molecules and the new second molecules,wherein each of the plurality of new first molecules and the plurality of new second molecules is generated by associating by a random manner each of a plurality of occurrences of a first target molecule and a second target molecule, respectively, in a sample with a label from a set of diverse labels, wherein the set of diverse labels comprises labels with m different label sequences,wherein each of the plurality of new first molecules and the plurality new second molecules comprises a copy of the first target molecule and a copy of the second target molecule, respectively, and a label sequence,wherein more than 90% of new first molecules of the plurality new first molecules and new second molecules of the plurality new second molecules have a label sequence that is different from the label sequences on the other new first molecules and the other new second molecules, respectively,wherein n1 and n2 are numbers of occurrences of the first target molecule and the second target molecule, respectively,wherein the ratio of the greater of n1 and n2 to m is smaller than 0.2, andwherein the numbers of the new first molecules and the new second molecules detected indicate the numbers of occurrences of the first target molecule and the second target molecule, respectively, in the sample.
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Abstract
Compositions, methods and kits are disclosed for high-sensitivity single molecule digital counting by the stochastic labeling of a collection of identical molecules by attachment of a diverse set of labels. Each copy of a molecule randomly chooses from a non-depleting reservoir of diverse labels. Detection may be by a variety of methods including hybridization based or sequencing. Molecules that would otherwise be identical in information content can be labeled to create a separately detectable product that is unique or approximately unique in a collection. This stochastic transformation relaxes the problem of counting molecules from one of locating and identifying identical molecules to a series of binary digital questions detecting whether preprogrammed labels are present. The methods may be used, for example, to estimate the number of separate molecules of a given type or types within a sample.
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Citations
28 Claims
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1. A method, comprising:
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detecting each of a plurality of new first molecules and a plurality of new second molecules by detecting the label sequence present on each of the new first molecules and the new second molecules, wherein each of the plurality of new first molecules and the plurality of new second molecules is generated by associating by a random manner each of a plurality of occurrences of a first target molecule and a second target molecule, respectively, in a sample with a label from a set of diverse labels, wherein the set of diverse labels comprises labels with m different label sequences, wherein each of the plurality of new first molecules and the plurality new second molecules comprises a copy of the first target molecule and a copy of the second target molecule, respectively, and a label sequence, wherein more than 90% of new first molecules of the plurality new first molecules and new second molecules of the plurality new second molecules have a label sequence that is different from the label sequences on the other new first molecules and the other new second molecules, respectively, wherein n1 and n2 are numbers of occurrences of the first target molecule and the second target molecule, respectively, wherein the ratio of the greater of n1 and n2 to m is smaller than 0.2, and wherein the numbers of the new first molecules and the new second molecules detected indicate the numbers of occurrences of the first target molecule and the second target molecule, respectively, in the sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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Specification