Digital counting of individual molecules by stochastic attachment of diverse labels

Patent metadata

Field	Value
Publication number	US-9290808-B2
Application number	US-201414540007-A
Country	US
Kind code	B2
Filing date	Nov 12, 2014
Priority date	Dec 15, 2009
Publication date	Mar 22, 2016
Grant date	Mar 22, 2016

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.

First claim

We claim:

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.

Assignees

• Cellular Res Inc

Inventors

• Fodor Stephen P A
• Fu Glenn K

Classifications

• C12N15/1065

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• G16B25/00

  ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6837

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title