DIGITAL COUNTING OF INDIVIDUAL MOLECULES BY STOCHASTIC ATTACHMENT OF DIVERSE LABELS
First Claim
1. A method, comprising:
- (a) attaching by a random manner to each of a plurality of occurrences of a first target molecule and a second target molecule in a sample a label from a set of diverse labels, wherein the set of diverse labels comprises labels with m different label sequences each having 6 or more nucleotides in length, thereby generating for each occurrence of the first target molecule and the second target molecule, a new first molecule and a new second molecule, respectively,wherein each of the new first molecules and the new second molecules comprise 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 the new first molecules and the 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, andwherein the ratio of the greater of n1 and n2 to m is smaller than 0.2;
(b) detecting each of the new first molecules and the new second molecules by detecting the label sequence present on each of the new first molecules and the new second molecules, 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.
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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
27 Claims
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1. A method, comprising:
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(a) attaching by a random manner to each of a plurality of occurrences of a first target molecule and a second target molecule in a sample a label from a set of diverse labels, wherein the set of diverse labels comprises labels with m different label sequences each having 6 or more nucleotides in length, thereby generating for each occurrence of the first target molecule and the second target molecule, a new first molecule and a new second molecule, respectively, wherein each of the new first molecules and the new second molecules comprise 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 the new first molecules and the 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, and wherein the ratio of the greater of n1 and n2 to m is smaller than 0.2; (b) detecting each of the new first molecules and the new second molecules by detecting the label sequence present on each of the new first molecules and the new second molecules, 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)
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Specification