Digital counting of individual molecules by stochastic attachment of diverse labels
View Patent ↗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.
1. A composition comprising a plurality of oligonucleotide labels, wherein each oligonucleotide label of the plurality of oligonucleotide labels comprises an oligo dT sequence, a sequencing primer binding site, a common sequence that is the same for all oligonucleotide labels of the plurality of oligonucleotide labels, and a unique label tag sequence, wherein the unique label tag sequence is selected from a set of at least m different label tag sequences.
2. The composition of claim 1 , further comprising a set of n target molecules hybridized to said plurality of oligonucleotide labels.
3. The composition of claim 2 , wherein the target molecules are RNA molecules.
4. The composition of claim 2 , wherein the target molecules are mRNA molecules.
5. The composition of claim 2 , wherein n has a value ranging from 1 to 100.
6. The composition of claim 2 , wherein n has a value ranging from 100 to 1,500.
7. The composition of claim 2 , wherein n has a value greater than 1,500.
8. The composition of claim 2 , wherein the target molecules comprise one or more different types of target molecules from a single cell.
9. The composition of claim 2 , wherein m is greater than n.
10. The composition of claim 9 , wherein a ratio m/n is greater than 5.
11. The composition of claim 1 , wherein m is between 100 and 1,000.
12. The composition of claim 1 , wherein m is between 1,000 and 10,000.
13. The composition of claim 1 , wherein the plurality of oligonucleotide labels are between 48 and 76 nucleotides in length.
14. A kit comprising a plurality of oligonucleotide labels, wherein each oligonucleotide label of the plurality of oligonucleotide labels comprises an oligo dT sequence, a sequencing primer binding site, a common sequence that is the same for all oligonucleotide labels of the plurality of oligonucleotide labels, and a unique label tag sequence, wherein the unique label tag sequence is selected from a set of at least m different label tag sequences.
15. The kit of claim 14 , wherein m is between 100 and 1,000.
16. The kit of claim 14 , wherein m is between 1,000 and 10,000.
17. The kit of claim 14 , wherein the plurality of oligonucleotide labels are between 48 and 76 nucleotides in length.
18. The kit of claim 14 , further comprising PCR amplification and sequencing reagents.