Digital counting of individual molecules by stochastic attachment of diverse labels

• US 10,047,394 B2
• Filed: 07/29/2016
• Issued: 08/14/2018
• 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:
  US15/224,460
• Publication Number:
  US 20160326584A1
• Time in Patent Office: 746 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:

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

wherein each of the new first molecules and the new second molecules comprise a copy of the first target molecule and a copy of the second target molecule, respectively, and a label,

wherein more than 90% of the new first molecules and the new second molecules have a label that is different from the labels on the other new first molecules and the new 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) detecting each of the new first molecules and the new second molecules by detecting the label present on each of the new first molecules and the new second molecules, wherein the numbers of the new first molecules and the new second molecules detected indicate the numbers of occurrences of the first molecule and the second molecule, respectively, in the sample.

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 m and n are related by E(k),

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 m and n are related by Var(k),

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 m and n are related by E(t),

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 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 labels present on the new first molecules comprises sequencing the new first molecules.

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

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

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

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

12. The method of claim 7, wherein the number of labels present on the new first molecules indicates the number of new first molecules.

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

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

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

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

17. The method of claim 1, wherein the first target molecule is mRNA.

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

19. The method of claim 1, wherein the first target molecule is DNA or RNA.

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