Digital counting of individual molecules by stochastic attachment of diverse labels

• US 10,059,991 B2
• Filed: 07/22/2016
• Issued: 08/28/2018
• 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.
• Primary Examiner(s): Mahatan, Channing S
• Application Number:
  US15/217,896
• Publication Number:
  US 20160376583A1
• Time in Patent Office: 767 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 for obtaining clonal populations derived from a first target molecule and a second target molecule in a sample comprising:

(a) attaching by a random manner to each of a plurality of occurrences of a first target molecule and a second target molecule a label from a set of diverse labels, wherein the set comprises labels with m different label sequences, thereby generating for each of the plurality of occurrences of the first target molecule and the second target molecule, a labeled first molecule and a labeled second molecule, respectively,

wherein each of the labeled first molecule and the labeled second molecule 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 labeled first molecules and the labeled second molecules have a label sequence that is different from the label sequences on the other labeled first molecules and the labeled second molecules, respectively,

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

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

(b) amplifying the labeled first molecules and the labeled second molecules to create a plurality of first clonal populations and a plurality of second clonal populations, respectively, wherein a first clonal population of the plurality of first clonal populations comprises copies of the first target molecule with a first identical label sequence, and wherein a second clonal population of the plurality of second clonal populations comprises copies of the second target molecule with a second identical label sequence; and

(c) detecting the label sequences present on the plurality of first clonal populations and the plurality of second clonal populations, thereby detecting the label sequences present on the labeled first molecules and the labeled second molecules.

2. The method of claim 1, wherein the labels attached to 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

3. The method of claim 1, wherein the labels attached to 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

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

wherein n is n₁ or n₂,

wherein

5. The method of claim 1, wherein the labels attached to 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

6. The method of claim 1, wherein detecting the label sequences present on the plurality of first clonal populations and the plurality of second clonal populations comprises sequencing the plurality of first clonal populations and the plurality of second clonal populations to obtain sequencing reads of the plurality of first clonal populations and the plurality of second clonal populations.

7. The method of claim 6, wherein sequencing the plurality of first clonal populations and the plurality of second clonal populations comprises mapping the sequencing reads to a reference sequence.

8. The method of claim 6, wherein sequencing the plurality of first clonal populations and the plurality of second clonal populations comprises aligning the sequencing reads.

9. The method of claim 6, wherein sequencing the plurality of first clonal populations and the plurality of second clonal populations comprises detecting a mismatch in the label or the occurrence of the first target molecule or the second molecule, respectively, in the sequencing reads of the first clonal population or the second clonal population, respectively.

10. The method of claim 6, wherein sequencing the plurality of first clonal populations and the plurality of second clonal populations comprises sequencing a portion of the label or the occurrence of the first target molecule and the second target molecule, respectively.

11. The method of claim 6, wherein sequencing the plurality of first clonal populations and the plurality of second clonal populations comprises generating a sequencing library from the plurality of first clonal populations and the plurality of second clonal populations.

12. The method of claim 1, comprising adding an adaptor to the labeled first molecules and the labeled second molecules.

13. The method of claim 12, wherein the adaptor comprises a binding site for a primer.

14. The method of claim 13, wherein the amplifying the labeled first molecules and the labeled second molecules comprises amplifying using the primer.

15. The method of claim 1, wherein the attaching of step (a) comprises ligation of the label to an occurrence of the target molecule or the second target molecule.

16. The method of claim 1, wherein the attaching of step (a) comprises reverse transcription of an occurrence of the first target molecule or the second target molecule.

17. The method of claim 1, wherein the attaching of step (a) comprises use of the label for primer extension.

18. The method of claim 1, wherein the attaching of step (a) occurs on one end of an occurrence of the first target molecule or the second target molecule.

19. The method of claim 1, wherein the attaching of step (a) occurs on both ends of an occurrence of the first target molecule or the second target molecule.

20. The method of claim 1, wherein the attaching of step (a) comprises reverse transcription of the plurality of occurrences of the first target molecule or the second target molecule.

21. The method of claim 1, wherein the plurality of occurrences of the first target molecule and the second target molecule is RNA.

22. The method of claim 1, wherein the plurality of occurrences of the first target molecule and the second target molecule is mRNA.

23. The method of claim 1, wherein the label of the set of diverse labels comprises an oligodT sequence.

24. The method of claim 23, wherein the label is attached to the occurrence of the first target molecule or the second target molecule by hybridization of the oligodT sequence of the label to a polyA tail of the occurrence of the first target molecule or the second target molecule.

25. The method of claim 1, wherein the plurality of occurrences of the first target molecule or the second target molecule is DNA.

26. The method of claim 1, further comprising fragmenting the plurality of occurrences of the first target molecule or the second target molecule.

27. The method of claim 26, wherein the plurality of occurrences of the first target molecule and the second target molecule is fragmented prior to the attaching of step (a).

28. The method of claim 26, wherein fragmenting the plurality of occurrences of the first target molecule and the second target molecule comprises restriction enzyme digestion of the plurality of occurrences of the first target molecule and the second target molecule.

29. The method of claim 1, wherein the amplifying comprises PCR.

30. The method of claim 1, wherein the amplifying comprises rolling circle amplification.

31. The method of claim 1, wherein the sample is a single cell.

32. The method of claim 1, wherein the sample is a cell population.

33. The method of claim 1, wherein the sample is a tissue.

34. The method of claim 1, wherein the copy of the first molecule comprises the complementary sequence or the reverse complementary sequence of the first molecule, or a portion of the complementary sequence or the reverse complementary sequence of the first molecule, and wherein the copy of the second molecule comprises the complementary sequence or the reverse complementary sequence of the second molecule, or a portion of the complementary sequence or the reverse complementary sequence of the second molecule.

35. The method of claim 1, wherein the copy of the first molecule comprises the sequence or a partial sequence of the first molecule, and wherein the copy of the second molecule comprises the sequence or a partial sequence of the second molecule.