Methods of identifying multiple epitopes in cells

Abstract

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 identifying whether a plurality of targets is present in a plurality of cells comprising:
 a) binding to the targets in the plurality of cells a plurality of tags, wherein a tag comprises a unique binding agent (UBA) conjugated to an anchor oligonudeotide;   b) annealing to the anchor a linker oligonudeotide to form a hybrid between the anchor and the linker so that the linker forms a 3′-overhang, and   c) assembling a cell originating barcode (COB) by subsequently adding multiple assayable polymer subunit (APS) oligonucleotides by a series of steps comprising:
 i annealing an APS to the 3′-overhang of the linker; 
 ii extending the linker by a polymerase thereby copying the annealed APS and forming an extended linker; 
 iii disassociating the annealed APS from the extended linker; and 
 iv repeating steps i-iii by adding APSs to the plurality of cells in an ordered manner during successive rounds of split pool synthesis wherein the APSs in each round anneal to the extended linkers and the linkers are further extended to create COBs that represent the identities of individual cells in which the tags are bound, and wherein the method does not include a step of isolating each cell in the plurality of cells. 
   
     
     
         2 . The method of  claim 1 , wherein the UBA is an antibody. 
     
     
         3 . The method of  claim 1 , wherein the linker comprises an epitope specific barcode (ESB) identifying the antibody target. 
     
     
         4 . The method of  claim 1 , wherein the UBA is a nucleic acid. 
     
     
         5 . The method of  claim 4 , wherein the nucleic acid is at least partially complementary to a target nucleic acid. 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein in the hybrid between the anchor and the linker, the linker further forms a 5′-overhang and after step b), the anchor is extended to copy the 5′-overhang of the linker. 
     
     
         8 . The method of  claim 7 , wherein the 5′-overhang of the linker comprises one or more modified nucleotides increasing stability of the hybrid. 
     
     
         9 . The method of  claim 8 , wherein the modified nucleotide is selected from of locked nucleic acid (LNA), peptide nucleic acid (PNA), AP-cytosine (G-clamp), 2-aminoadenine and propynyl pyrimidine. 
     
     
         10 . The method of  claim 1 , wherein the annealed APS is disassociated from the extended linker by contacting the sample with a duplex destabilizing agent selected from alkali, formamide, sulfolane, ethylene carbonate, pyrrolidone and a primary amide. 
     
     
         11 . The method of  claim 10 , wherein the duplex destabilizing agent is added at a concentration sufficient to dissociate the APS but not sufficient to dissociated the hybrid between the anchor and the extended linker. 
     
     
         12 . The method of  claim 1 , wherein the polymerase lacks 3′-5′ proof-reading activity. 
     
     
         13 . The method of  claim 1 , wherein the polymerase is a Y-family polymerase. 
     
     
         14 . The method of  claim 1 , wherein the polymerase is an error prone polymerase. 
     
     
         15 . The method of  claim 1 , wherein the polymerase is Taq polymerase in the presence of manganese ions. 
     
     
         16 . The method of  claim 1 , wherein the APS comprises a unique barcode. 
     
     
         17 . The method of  claim 1 , wherein the APS comprises at least one annealing region capable of forming a duplex with the linker or the extended linker. 
     
     
         18 . The method of  claim 1 , further comprising a step of sequencing the cell originating barcode (COB). 
     
     
         19 . The method of  claim 18 , wherein sequencing includes sequencing the entire extended linker. 
     
     
         20 . The method of  claim 1 , wherein the linker comprises a primer binding site. 
     
     
         21 . The method of  claim 20 , wherein the primer binding site is in the 5′-overhang of the linker. 
     
     
         22 . The method of  claim 1 , wherein at least one APS comprises a primer binding site. 
     
     
         23 . The method of  claim 22 , wherein the APS comprising the primer binding site is added in the final round of split-pool synthesis. 
     
     
         24 . A composition comprising a plurality of cells, wherein within each cell, at least one target is bound to a tag comprising a target-specific unique binding agent (UBA) conjugated to an anchor oligonudeotide annealed to an extended linker oligonudeotide wherein a 3′-overhang of the extended linker comprises a unique cell originating barcode (COB) consisting of copies of multiple assayable polymer subunit (APS). 
     
     
         25 . The composition of  claim 24 , wherein the anchor annealed to the extended linker is an extended anchor formed by extending a 3′-end of the anchor to copy a 5′ overhang of the linker. 
     
     
         26 . The composition of  claim 24 , wherein the COB comprises unique barcodes copied from multiple APSs. 
     
     
         27 . The composition of  claim 24 , wherein the extended linker comprises at least one primer binding site. 
     
     
         28 . A method for identifying whether a plurality of targets is present in a plurality of cells comprising:
 a) binding to the targets in the plurality of cells a plurality of tags, wherein a tag comprises a target-specific unique binding agent (UBA) conjugated to an anchor oligonudeotide;   b) annealing to the anchor a linker oligonudeotide to form a hybrid between the anchor and the linker so that the anchor forms a 3′-overhang, and   c) assembling a cell originating barcode (COB) by subsequently adding multiple assayable polymer subunit (APS) oligonucleotides by a series of steps comprising:
 i annealing an APS to the 3′-overhang of the anchor; 
 ii extending the anchor by a polymerase thereby copying the annealed APS and forming an extended anchor; 
 iii disassociating the annealed APS from the extended anchor; and 
   
       repeating steps i-iii by adding APSs to the plurality of cells in an ordered manner during successive rounds of split pool synthesis wherein the APSs in each round anneal to the extended anchors and the anchors are further extended to create COBs that represent the identities of individual cells in which the tags are bound, and wherein the method does not include a step of isolating each cell in the plurality of cells.