METHODS AND COMPOSITIONS FOR LABELING TARGETS

• US 20160257993A1
• Filed: 02/26/2016
• Published: 09/08/2016
• Est. Priority Date: 02/27/2015
• Status: Active Application

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Wilhelmy, Julie, Simbirsky, Maria, Shum, Eleen, Fu, Glenn
• Application Number:
  US15/055,407
• Publication Number:
  US 20160257993A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

Embodiments provided herein relate to methods and compositions for labeling a nucleic acid in a sample with a stochastic barcode. Some embodiments relate to methods and compositions for characterizing a sample by identifying the TCR alpha chain or beta chain of a T cell.

Claims

1. A method of labeling a nucleic acid in a sample, comprising:

hybridizing a target with a first oligonucleotide comprising a universal adaptor sequence and a target-specific sequence;

extending said first oligonucleotide to generate a first nucleotide sequence;

hybridizing said first nucleotide sequence with a second oligonucleotide;

extending said second oligonucleotide to generate a second nucleotide sequence;

hybridizing said second nucleotide sequence with a third oligonucleotide comprising a binding site for a first amplification primer and the universal adaptor sequence, wherein one or both of the first oligonucleotide and the third oligonucleotide comprises part of a stochastic barcode;

extending said third oligonucleotide to generate a third nucleotide sequence; and

amplifying said third nucleotide sequence using a first amplification primer to generate a plurality of a fourth nucleotide sequences,

wherein each of the plurality of the fourth nucleotide sequences comprises said target and the stochastic barcode.

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

3. The method of claim 1, wherein the target is an mRNA molecule.

4. The method of claim 3, wherein the mRNA molecule encodes a T cell receptor (TCR) alpha chain or beta chain.

5. The method of claim 4, wherein the target-specific sequence specifically binds to the C region of the TCR alpha chain or beta chain.

6. The method of claim 1, wherein the stochastic barcode comprises a molecular label.

7. The method of claim 1, wherein the sample comprises a plurality of the target.

8. The method of claim 7, wherein each of the plurality of the target is labeled with a distinct stochastic barcode.

9. The method of claim 8, comprising counting the number of the plurality of the target by counting the distinct stochastic barcodes associated with the target.

10. A method of labeling a target in a sample for quantification of said target, comprising:

hybridizing said target with a first oligonucleotide comprising a first universal adaptor sequence and a target-specific sequence;

extending said first oligonucleotide to generate a first nucleotide sequence;

hybridizing said first nucleotide sequence with a second oligonucleotide comprising a second universal adaptor sequence and a second target-specific sequence;

extending said second oligonucleotide to generate a second nucleotide sequence;

hybridizing said second nucleotide sequence with a third oligonucleotide comprising a binding site for a first amplification primer and the first universal adaptor sequence;

extending said third oligonucleotide to generate a third nucleotide sequence;

hybridizing said third nucleotide sequence with a fourth oligonucleotide comprising a binding site for a second amplification primer and the second universal adaptor sequence, wherein one or both of the third oligonucleotide and the fourth oligonucleotide comprises part of a stochastic barcode;

extending said fourth oligonucleotide to generate a fourth nucleotide sequence; and

amplifying said fourth nucleotide sequence using a first amplification primer and a second amplification primer to generate a plurality of a fifth nucleotide sequences,

wherein each of the plurality of the fifth nucleotide sequences comprises said target and the stochastic barcode.

11. The method of claim 10, wherein the mRNA molecule encodes a T cell receptor (TCR) alpha chain or beta chain.

12. The method of claim 11, wherein the target-specific sequence specifically binds to the C region of the TCR alpha chain or beta chain.

13. The method of claim 10, wherein the second target-specific sequence specifically binds to a region selected from the group consisting of:

a V region of a TCR alpha chain or beta chain, the junction of a V and D region of a TCR alpha chain or beta chain, or any combination thereof.

14. The method of claim 10, wherein the stochastic barcode comprises a molecular label.

15. A method of labeling a target in a sample for quantification of said target, comprising:

hybridizing said target with a first oligonucleotide comprising a target-specific sequence and a binding site for a first amplification primer;

extending said first oligonucleotide to generate a first nucleotide sequence;

hybridizing said first nucleotide sequence with a second oligonucleotide comprising a universal adaptor sequence and a second target-specific sequence;

extending said second oligonucleotide to generate a second nucleotide sequence;

hybridizing said second nucleotide sequence with a third oligonucleotide comprising a binding site for a second amplification primer and a sequence that specifically binds the universal adaptor sequence, wherein one or both of the second oligonucleotide and the third oligonucleotide comprises part of a stochastic barcode;

extending said third oligonucleotide to generate a third nucleotide sequence; and

amplifying said third nucleotide sequence using a first amplification primer and a second amplification primer to generate a plurality of a fourth nucleotide sequences,

wherein each of the plurality of the fourth nucleotide sequences comprises said target and the stochastic barcode.

16. The method of claim 15, wherein the sample is a single cell.

17. The method of claim 15, wherein the target is an mRNA molecule.

18. The method of claim 17, wherein the mRNA molecule encodes a T cell receptor (TCR) alpha chain or beta chain.

19. The method of claim 18, wherein the target-specific sequence specifically binds to the C region of the TCR alpha chain or beta chain.

20. The method of claim 15, wherein the second target-specific sequence specifically binds to a region selected from the group consisting of:

a V region of a TCR alpha chain or beta chain, the junction of a V and D region of a TCR alpha chain or beta chain, or any combination thereof.

21. A method of characterizing a sample, comprising:

hybridizing a first target in the sample with a first oligonucleotide comprising a first target-specific sequence and a first stochastic barcode, wherein the first target is an mRNA encoding a TCR alpha chain or beta chain;

hybridizing a second target in the sample with a second oligonucleotide comprising a second target-specific sequence and a second stochastic barcode;

extending the first oligonucleotide and the second oligonucleotide to incorporate the first stochastic barcode into the first target and the second stochastic barcode into the second target; and

identifying the sequences of the first target and the second target.

22. The method of claim 21, wherein the sample is a single cell.

23. The method of claim 21, wherein the first target-specific sequence specifically binds to the C region of the TCR alpha chain or beta chain.

24. The method of claim 21, wherein the extending comprises amplifying the first target with a forward primer.

25. The method of claim 24, wherein the forward primer specifically binds to a region selected from the group consisting of:

a V region of a TCR alpha chain or beta chain, the junction of a V and D region of a TCR alpha chain or beta chain, or any combination thereof.

26. The method of claim 21, wherein the second target is selected from the group consisting of the genes listed in FIG. 39.