DIGITAL COUNTING OF INDIVIDUAL MOLECULES BY STOCHASTIC ATTACHMENT OF DIVERSE LABELS

• US 20150099661A1
• Filed: 11/12/2014
• Published: 04/09/2015
• Est. Priority Date: 12/15/2009
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: FODOR, STEPHEN P.A., FU, GLENN K.
• Granted Patent: US 9,290,808 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/10 Gene or protein expression profiling; Expression-ratio estimation or normalisation
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids

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.

Claims

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 label-tags; and

ii) generating a plurality of target-label-tag molecules by hybridizing label-tag molecules of the plurality of label-tag molecules 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 the 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 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 the 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 a poly T) 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 molecules to target molecules is performed under stringent conditions.