Digital counting of individual molecules by stochastic attachment of diverse labels
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 method for determining a number of occurrences of a target molecule from a single cell, the method comprising:
a) generating an indexed library by performing a labeling step, the labeling step comprising:
i) combining in a specified container a sample comprising a plurality of target molecules from the single cell with a plurality of diverse label-tag, wherein the ratio of the number of diverse label-tag sequences to the number of occurrences of a target molecule is greater than 5; and
ii) generating a plurality of target-label-tag molecules by hybridizing label-tags of the plurality of diverse label-tag to target molecules of the plurality of target molecules and performing an extension reaction, wherein a target-label-tag molecule comprises a label-tag and a portion of a complementary sequence of a target molecule;
b) amplifying the target-label-tag molecules from the indexed library; and
c) sequencing at least a portion of the amplified product of step (b) to determine a number of different label-tag sequences associated with the portion of the complementary sequence of a target molecule from the single cell, wherein the number of different label-tag sequences indicates the number of occurrences of the target molecule from the single cell.
2. The method of claim 1 , wherein the sequencing of step (c) results in detection of the portion of the complementary sequence of the target molecule.
3. The method of claim 1 , wherein the method is multiplexed.
4. The method of claim 1 , wherein the plurality of target molecules comprise nucleic acid molecules.
5. The method of claim 4 , wherein the nucleic acid molecules comprise genomic DNA.
6. The method of claim 4 , wherein the nucleic acid molecules comprise cDNA.
7. The method of claim 4 , wherein the nucleic acid molecules comprise RNA.
8. The method of claim 7 , wherein the RNA is mRNA.
9. The method of claim 1 , wherein a target molecule from the single cell comprises a polymorphism.
10. The method of claim 9 , wherein the polymorphism is a single nucleotide polymorphism.
11. The method of claim 9 , wherein the polymorphism is an indel.
12. The method of claim 1 , wherein the specified container comprises a well.
13. The method of claim 1 , wherein the plurality of label-tags are attached to a bead.
14. The method of claim 1 , wherein a label-tag of the plurality of label-tags further comprises at least one universal primer.
15. The method of claim 1 , wherein each label-tag of the plurality of label-tags further comprises a common sequence.
16. The method of claim 1 , wherein a label-tag of the plurality of label-tags further comprises a target-specific recognition sequence.
17. The method of claim 1 , wherein a label-tag of the plurality of label-tags further comprises an oligo dT sequence.
18. The method of claim 1 , wherein the plurality of label-tags comprises two or more target-specific recognition sequences.
19. The method of claim 1 , wherein the plurality of label-tags comprises between 100 and 10,000 different label-tag sequences.
20. The method of claim 1 , wherein amplifying comprises performing a polymerase chain reaction.
21. The method of claim 1 , further comprising storing the indexed library.
22. The method of claim 1 , further comprising performing the steps of amplifying and sequencing multiple times.
23. The method of claim 1 , wherein the hybridization is stochastic.
24. The method of claim 1 , wherein the sequencing step comprises determining a genotype.
25. The method of claim 3 , wherein the sequencing step comprises determining a gene expression profile.
26. The method of claim 1 , wherein the single cell is a human cell.
27. The method of claim 1 , wherein the single cell is a tumor cell.
28. The method of claim 1 , wherein the single cell is a bacterium.
29. The method of claim 1 , wherein the hybridization of the label-tag to target molecules is performed under stringent conditions.