Increasing dynamic range for identifying multiple epitopes in cells
The invention provides methods, compositions, kits and devices for the detection of target molecules. In some embodiments, the invention allows for multiplexed target molecule detection.
1. A method for detecting target molecules in a sample comprising:
(a) obtaining:
(i) a population of cells comprising the target molecules,
(ii) a plurality of target specific unique binding agents (UBA)s,
(iii) a plurality of epitope specific barcode (ESB) mixtures, each capable of associating with the UBAs, wherein at least one ESB mixture comprises labeled and unlabeled ESBs at a predetermined ratio, and
(iv) a plurality of round-specific assayable polymer subunit (APS) sets, each set comprising a plurality of APSs that are detectably distinct from each other;
(b) contacting the UBAs with said population of cells;
(c) contacting the plurality of ESBs with said population of cells wherein the labeled ESBs and the unlabeled ESBs bind to the UBAs; and
(d) conducting split pool synthesis of cell originating barcodes (COBs) from the plurality of APSs sets by a method comprising
(i) splitting the mixture into two or more samples;
(ii) adding one APS from an APS set specific for the round per sample to the two or more samples from step (i), where a complex is formed with the least one target molecule, the UBA, the ESB, and a first APS;
(iii) pooling the two or more samples from step (i) into one sample;
(iv) splitting the sample from step (iii) into two or more samples;
(v) adding one APS from an APS set specific for the round per sample to the two or more samples from step (iv), where a complex is formed with the least one target molecule, the UBA, the ESB, the first APS, and the second APS; and optionally, repeating steps (iii) through (v) one or more times;
wherein APSs comprise an annealing region and the annealing region of an added APS is complementary to an available annealing region of an APS from a previous round and wherein APSs sets in each round comprise a round-specific subcode so that the synthesized COB comprises a unique label code associated with the target molecule and the cell in the mixture.
2. The method of claim 1 , further comprising obtaining at least one target specific signal by detecting at least part of said UBA-ESB-APS complex.
3. The method of claim 2 , further comprising quantifying the target molecule associated with the target specific signal by normalizing the target specific signal with a multiplier, wherein the multiplier is based on the predetermined ratio.
4. The method of claim 2 , wherein the detecting comprises sequencing.
5. The method of claim 3 , wherein the multiplier is the inverse of the predetermined ratio.
6. The method of claim 1 , wherein each target is mRNA.
7. The method of claim 1 , wherein the UBA is a nucleic acid.
8. The method of claim 1 , wherein the UBA comprises an antibody.
9. The method of claim 1 , wherein the ESB comprises a common linker (CL).
10. The method of claim 1 , wherein the ESB codes the target identity.
11. The method of claim 1 , wherein the ESB comprises a nucleic acid.
12. The method of claim 1 , wherein the APS comprises a nucleic acid.
13. The method of claim 1 , wherein the APSs, an ESB, and a UBA are linked by ligation.
14. The method of claim 1 , wherein the APSs, the ESB, and the UBA are capable of being linked using Click chemistry.
15. The method of claim 1 , wherein the APS or the ESB comprises an amplification primer binding region.