Methods for determining a location of a biological analyte in a biological sample

Abstract

Provided herein are methods of determining a location of a biological analyte in a biological sample.

What is claimed is: 
     
         1 . A method for determining a location of an analyte within a region of interest in a biological sample, the method comprising:
 (a) contacting a cell labelling agent with the biological sample, wherein the cell labeling agent binds specifically to the analyte and comprises a molecular tag barcode;   (b) contacting the biological sample with a substrate, wherein the substrate comprises a plurality of capture probes, wherein a capture probe comprises a capture domain that specifically binds the molecular tag barcode or a portion thereof;   (c) identifying the region of interest within the biological sample; and   (d) detecting the presence of the molecular tag barcode within the region of interest, thereby determining the location of the analyte within the biological sample.   
     
     
         2 . The method of  claim 1 , wherein the molecular tag barcode further comprises a cleavage domain. 
     
     
         3 . The method of  claim 2 , wherein the cleavage domain is cleavable by at least one of light, a chemical molecule, and an enzyme. 
     
     
         4 . The method of  claim 2 , wherein the cleavage domain is cleaved by photolysis. 
     
     
         5 . The method of  claim 1 , wherein the analyte comprises RNA. 
     
     
         6 . The method of  claim 1 , wherein the analyte comprises DNA. 
     
     
         7 . The method of  claim 1 , wherein the analyte comprises a protein. 
     
     
         8 . The method of  claim 1 , wherein the analyte comprises a cell surface analyte, an intracellular analyte, an extracellular analyte, or combinations thereof. 
     
     
         9 . The method of  claim 1 , wherein the cell labelling agent comprises an antibody or an antigen-binding fragment thereof. 
     
     
         10 . The method of  claim 1 , wherein the cell labelling agent comprises a lipophilic molecule. 
     
     
         11 . The method of  claim 1 , wherein the capture domain hybridizes to the molecular tag barcode or a portion thereof. 
     
     
         12 . The method of  claim 11 , wherein the molecular tag barcode identifies the cell labelling agent. 
     
     
         13 . The method of  claim 1 , wherein an electric field is applied to the biological sample and the substrate. 
     
     
         14 . The method of  claim 1 , further comprising, prior to step (d), removing the biological sample from the substrate. 
     
     
         15 . The method of  claim 1 , wherein the biological sample is treated with a permeabilization reagent prior to or after contacting the substrate with the biological sample. 
     
     
         16 . The method of  claim 1 , wherein the plurality of capture probes comprise identical capture domains. 
     
     
         17 . The method of  claim 1 , wherein the plurality of capture probes comprise two or more non-identical capture domains. 
     
     
         18 . The method of  claim 1 , wherein the plurality of capture probes are directly attached to the substrate. 
     
     
         19 . The method of  claim 1 , wherein the plurality of capture probes are indirectly attached to the substrate. 
     
     
         20 . The method of  claim 19 , wherein one or more features are attached to the substrate. 
     
     
         21 . The method of  claim 20 , wherein the plurality of capture probes are attached to the one or more features. 
     
     
         22 . The method of  claim 20 , wherein the one or more features comprises a bead. 
     
     
         23 . The method of  claim 22 , wherein the bead is a gel bead. 
     
     
         24 . The method of  claim 1 , wherein the detecting in step (d) comprises performing sequencing on the substrate. 
     
     
         25 . The method of  claim 24 , wherein the sequencing on the substrate comprises one or more cycles of hybridization with labelled probes. 
     
     
         26 . The method of  claim 25 , wherein the labelled probes are labelled with at least one of a fluorescent molecule, a fluorescent protein, a radioactive molecule, or an enzyme. 
     
     
         27 . The method of  claim 1 , wherein the capture domain comprises an oligo(dT) sequence. 
     
     
         28 . The method of  claim 27 , wherein the capture domain hybridizes to a poly(A) sequence of an mRNA, the molecular tag barcode or a portion thereof, or both. 
     
     
         29 . The method of  claim 1 , wherein the substrate is located in a flow cell and a set of fluorescent probes are hybridized to the molecular tag barcode. 
     
     
         30 . The method of  claim 29 , wherein the set of fluorescent probes are imaged, the substrate is heated, and the set of fluorescent probes are washed out of the flow cell.