DIGITAL COUNTING OF INDIVIDUAL MOLECULES BY STOCHASTIC ATTACHMENT OF DIVERSE LABELS

• US 20150099673A1
• 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 9,708,659 B2
• Time in Patent Office: Days
• US Class Current: 506/37
• 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 system for counting n, wherein n is a number of nucleic acid target molecules that are present in a sample, the system comprising:

a a diverse set of labels, wherein the set comprises different labels, wherein >

n; and

b a plurality of reaction vessels for attaching a label from the diverse set of labels to each occurrence of the nucleic acid target molecules from a single cell to generate a set of labeled nucleic acid target molecules, wherein the individual labeled nucleic acid target molecules comprise all or a portion of the complementary sequence of a nucleic acid target molecule and a label from the diverse set of labels.

2. The system of claim 1, further comprising a processor for calculating n from a number of labeled nucleic acid target molecules detected using Poisson statistics.

3. The system of claim 2, wherein the number of labeled nucleic acid target molecules is detected by nucleic acid sequencing.

4. The system of claim 1, wherein the nucleic acid target molecules present in the sample are DNA molecules, and wherein the labeled nucleic acid target molecules comprise complementary sequences of the DNA molecule sequences attached to a label from the diverse set of labels.

5. The system of claim 1, wherein the nucleic acid target molecules present in the sample are mRNA molecules, and wherein the labeled nucleic acid target molecules comprise cDNA complementary sequences of the mRNA molecule sequences attached to a label from the diverse set of labels.

6. The system of claim 1, wherein the ratio of to n is greater than 5.

7. The system of claim 1, wherein the ratio of to n is greater than 100.

8. The system of claim 1, wherein the diverse set of labels comprise a plurality of oligonucleotides.

9. The system of claim 8, wherein the plurality of oligonucleotides are attached to one or more beads.

10. The system of claim 9, wherein each oligonucleotide of the plurality of oligonucleotides comprises a common sequence, a variable target label sequence, and a target-specific recognition sequence capable of attaching to or hybridizing to the target molecule.

11. The system of claim 10, wherein the oligonucleotides further comprise one or more primer sequences.

12. The system of claim 11, wherein at least a portion of the plurality of reaction vessels contain a single bead.

13. The system of claim 12, wherein the plurality of reaction vessels that contain a single bead also contain a single cell.

14. The system of claim 13, wherein the plurality of reaction vessels are microwells.

15. An apparatus for stochastically labeling target molecules in single cells, the apparatus comprising:

a a substrate comprising a plurality of microwells;

b a plurality of beads comprising a plurality of diverse oligonucleotide labels; and

c a sample preparation unit configured to transfer all or a portion of a sample comprising cells into the microwells.

16. The apparatus of claim 15, wherein the plurality of diverse oligonucleotides labels comprise a common sequence, a set of variable target label sequences, and a target specific recognition sequence capable of attaching to or hybridizing to a nucleic acid target molecule.

17. The apparatus of claim 16, wherein the plurality of diverse oligonucleotides further comprise one or more primer sequences.

18. The apparatus of claim 15, wherein the plurality of beads are magnetic beads.

19. The apparatus of claim 18, wherein the sample preparation unit further comprises a magnet for moving the magnetic beads into or out of the microwells.

20. The apparatus of claim 15, wherein at least a portion of the microwells contain a single bead.

21. The apparatus of claim 20, wherein at least a portion of the microwells that contain a single bead also contain a single cell for determination of a number of transcripts from the single cell.

22. The apparatus of claim 21, wherein a label from the plurality of diverse oligonucleotide labels is attached to each occurrence of a target molecule in the single cell in a stochastic manner, thereby generating a set of labeled nucleic acid target molecules that each comprises at least a portion of the sequence of the nucleic acid target molecule and a label.

23. The apparatus of claim 15, wherein the sample preparation unit further comprises a thermal cycling mechanism configured to perform PCR amplification.

24. The apparatus of claim 15, further comprising an optical detection unit.

25. The apparatus of claim 15, further comprising a processor for analyzing data collected by the optical detection unit.

26. The apparatus of claim 25, wherein the processor is configured to for calculating n from a number of labeled nucleic acid target molecules detected using Poisson statistics.