Digital counting of individual molecules by stochastic attachment of diverse labels

• US 10,202,646 B2
• Filed: 07/18/2018
• Issued: 02/12/2019
• Est. Priority Date: 12/15/2009
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Fodor, Stephen P. A., Fu, Glenn K.
• Primary Examiner(s): Mahatan, Channing S
• Application Number:
  US16/038,832
• Publication Number:
  US 20180327836A1
• Time in Patent Office: 209 Days
• Field of Search: None
• 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, comprising:

obtaining a plurality of first molecules and a plurality of second molecules,

wherein each of the plurality of first molecules and the plurality of second molecules is generated by associating by a random manner each of occurrences of a first target molecule and occurrences of a second target molecule, respectively, with a label from a set of diverse labels,

wherein the set of diverse labels comprises labels with m different label sequences,

wherein each of the plurality of first molecules and the plurality second molecules comprises a copy of the first target molecule and a copy of the second target molecule, respectively, and a label sequence,

wherein more than 90% of the plurality of first molecules and the plurality of second molecules have a label sequence that is different from the label sequences on the other of the plurality of first molecules and the other of the plurality of second molecules, respectively,

wherein n₁ and n₂ are a number of occurrences of the first target molecule and a number of occurrences the second target molecule, respectively, and

wherein the ratio of the greater of n₁ and n₂ to m is smaller than 0.2; and

detecting each of the plurality of first molecules and the plurality of second molecules by detecting the label sequence present on each of the plurality of first molecules and the plurality of second molecules, respectively,

wherein the detected first molecules and the detected second molecules indicate the number of occurrences of the first target molecule and the number of occurrences of the second target molecule, respectively.

2. The method of claim 1, wherein the labels associated with the occurrences of the first target molecule or the second target molecule comprise labels with k different label sequences,

wherein n is n₁ or n₂,

wherein m and n are related by E(k),

wherein

3. The method of claim 1, wherein the labels associated with the occurrences of the first target molecule or the second target molecule comprise labels with k different label sequences,

wherein n is n₁ or n₂,

wherein m and n are related by Var(k),

wherein

4. The method of claim 1, wherein labels with t different label sequences are associated with the occurrences of the first target molecule or the second molecule exact x times,

wherein n is n₁ or n₂,

wherein m and n are related by E(t),

wherein

5. The method of claim 1, wherein the labels associated with the occurrences of the first target molecule or the second target molecule comprise labels with k different label sequences,

wherein n is n₁ or n₂,

wherein m and n are related by Var(k),

wherein

6. The method of claim 1, wherein the ratio of the greater of n₁ and n₂ to m is smaller than 0.02.

7. The method of claim 1, wherein detecting the label sequences present on the plurality of first molecules comprises sequencing the plurality of first molecules.

8. The method of claim 7, further comprising mapping sequencing reads that are obtained from sequencing the plurality of first molecules to a reference sequence.

9. The method of claim 7, further comprising aligning sequencing reads obtained from sequencing the plurality of first molecules.

10. The method of claim 7, further comprising detecting a mismatch in the label sequence or the copy of the first target molecule in sequencing reads obtained from sequencing the plurality of first molecules.

11. The method of claim 7, wherein sequencing the plurality of first molecules comprises sequencing a portion of the label sequence or a portion of the copy of the first target molecule.

12. The method of claim 7, wherein the label sequences present on the plurality of first molecules indicates the number of occurrences of the first target molecule.

13. The method of claim 12, comprising scoring a label sequence in sequencing reads obtained from sequencing the first molecules.

14. The method of claim 1, comprising adding an adaptor to each of the occurrences of the first target molecule.

15. The method of claim 1, wherein the obtaining step comprises associating by a random manner the occurrences of the first target molecule and the occurrences of the second target molecule with labels from the set of diverse labels to generate the plurality of first molecules and the plurality of second molecules, respectively.

16. The method of claim 15, wherein the associating step comprises reverse transcription of an occurrence of the first target molecule.

17. The method of claim 15, wherein the associating step comprises reverse transcription of the occurrences of the first target molecule.

18. The method of claim 15, wherein the associating step is performed using a reverse transcriptase, a polymerase, or a combination thereof.

19. The method of claim 1, wherein the first target molecule comprises an oligonucleotide.

20. The method of claim 19, wherein the oligonucleotide is ribonucleic acid (RNA).

21. The method of claim 19, wherein the oligonucleotide is deoxyribonucleic acid (DNA).

22. The method of claim 1, wherein the first target molecule comprises ribonucleic acid (RNA), and wherein the second target molecule comprises deoxyribonucleic acid (DNA).

23. The method of claim 1, wherein one or more of the first target molecule and the second target molecule comprise a ribonucleic acid (RNA), deoxyribonucleic acid (DNA), an antibody, or a combination thereof.

24. The method of claim 1, wherein the first target molecule is associated with an antibody.

25. The method of claim 1, wherein the first target molecule and/or the second target molecule comprise an oligonucleotide-labeled antibody.

26. The method of claim 25, wherein the oligonucleotides of the first target molecule and the second target molecules have different sequences.

27. The method of claim 1, wherein the label from the set of diverse labels comprises an oligo-dT sequence.

28. The method of claim 1, wherein the occurrences of the first target molecule and the occurrences of the second target molecule are from a single cell.

29. The method of claim 1, wherein the occurrences of the first target molecule are from a first sample, and wherein the occurrences of the second target molecule are from a second sample.

30. The method of claim 1, wherein the obtaining step comprises receiving the plurality of first molecules and the plurality of second molecules.