METHODS AND COMPOSITIONS FOR LABELING TARGETS
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.
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 .