Multiomic analysis device and methods of use thereof

Abstract

Disclosed herein, inter alia, are devices, compositions, kits, and methods for interrogating biological samples.

1 .- 43 . (canceled) 
     
     
         44 . A method of sequencing a plurality of target nucleic acids of a cell in situ, said method comprising the following steps in situ for each of the plurality of target nucleic acids:
 hybridizing an oligonucleotide primer to the target nucleic acid, wherein said target nucleic acid is on a microplate;   circularizing the oligonucleotide primer to generate a circular oligonucleotide; amplifying the circular oligonucleotide by extending an amplification primer hybridized to the circular oligonucleotide with a strand-displacing polymerase; wherein the amplification primer extension generates an extension product comprising multiple complements of the circular oligonucleotide; and sequencing the extension product,   wherein said microplate is coupled to a device comprising:   a sample stage configured to be coupled to a microplate receiver;   a microplate receiver configured to be coupled to the microplate;   at least one heating element thermally coupled to the microplate receiver;   a fluidics dispenser configured to dispense one or more reagents into the microplate;   an imaging system configured to detect one or more features in the microplate; and   a structure physically coupled to the sample stage, the heating element, the fluidics dispenser, and the imaging system.   
     
     
         45 . The method of  claim 44 , wherein circularizing comprises extending the 3′ end of the oligonucleotide primer along the target nucleic acid to generate a complementary sequence, and ligating the complementary sequence to the 5′ end of the oligonucleotide primer. 
     
     
         46 . A method of detecting a plurality of different targets within an optically resolved volume of a cell in situ, wherein the targets are nucleic acid sequences or proteins on a microplate; said method comprising:
 associating a different oligonucleotide barcode from a known set of barcodes with each of the plurality of targets;   sequencing each barcode to obtain a multiplexed signal in the cell in situ;   demultiplexing the multiplexed signal by comparison with the known set of barcodes; and   detecting the plurality of targets by identifying the associated barcodes detected in the cell,   wherein said microplate is coupled to a device comprising:   a sample stage configured to be coupled to a microplate receiver;   a microplate receiver configured to be coupled to the microplate;   at least one heating element thermally coupled to the microplate receiver;   a fluidics dispenser configured to dispense one or more reagents into the microplate;   an imaging system configured to detect one or more features in the microplate; and   a structure physically coupled to the sample stage, the heating element, the fluidics dispenser, and the imaging system.   
     
     
         47 .- 49 . (canceled) 
     
     
         50 . The method of  claim 44 , wherein the device comprises at least one structure configured to enclose a region and control temperature within said region. 
     
     
         51 . The method of  claim 50 , wherein the device is configured to perform temperature cycling of the microplate. 
     
     
         52 . The method of  claim 44 , wherein the device comprises at least one manifold coupled to the structure, wherein the at least one manifold comprises at least one of a reagent aspiration manifold and a reagent dispense manifold. 
     
     
         53 . The method of  claim 44 , wherein the device comprises an integrated system of one or more interconnected chambers, ports, and channels in fluid communication and configured for carrying out an analytical reaction or process, either alone or in cooperation with an appliance or instrument that provides support functions. 
     
     
         54 . The method of  claim 44 , wherein the microplate comprises 4, 6, 12, 24, 48, 96, 384 or 1536 sample wells. 
     
     
         55 . The method of  claim 44 , wherein the microplate comprises glass. 
     
     
         56 . The method of  claim 44 , wherein the target nucleic acid comprises RNA. 
     
     
         57 . The method of  claim 44 , wherein the target nucleic acid comprises cDNA. 
     
     
         58 . The method of  claim 46 , wherein the targets are nucleic acid sequences. 
     
     
         59 . The method of  claim 58 , wherein associating an oligonucleotide barcode with each of the plurality of targets comprises hybridizing a padlock probe to two adjacent nucleic acid sequences of the target, wherein the padlock probe is a single-stranded polynucleotide having a 5′ and a 3′ end, the padlock probe comprises at least one oligonucleotide barcode, and wherein the padlock probe comprises a primer binding sequence from a known set of primer binding sequences. 
     
     
         60 . The method of  claim 46 , wherein the targets are proteins. 
     
     
         61 . The method of  claim 60 , wherein associating an oligonucleotide barcode with each of the plurality of targets comprises contacting each of the targets with a specific binding reagent, wherein the specific binding reagent comprises an oligonucleotide barcode, hybridizing a padlock probe to two adjacent nucleic acid sequences of the barcode, wherein the padlock probe is a single-stranded polynucleotide having a 5′ and a 3′ end, and wherein the padlock probe comprises a primer binding sequence from a known set of primer binding sequences. 
     
     
         62 . The method of  claim 61 , wherein the specific binding reagent comprises an antibody, single-chain Fv fragment (scFv), antibody fragment-antigen binding (Fab), or an aptamer. 
     
     
         63 . The method of  claim 44 , wherein sequencing comprises sequencing-by-synthesis, sequencing by ligation, sequencing-by-hybridization, or pyrosequencing, and sequencing generates a sequencing read. 
     
     
         64 . The method of  claim 46 , wherein sequencing comprises sequencing-by-synthesis, sequencing by ligation, sequencing-by-hybridization, or pyrosequencing, and sequencing generates a sequencing read. 
     
     
         65 . The method of  claim 46 , wherein the microplate comprises glass. 
     
     
         66 . The method of  claim 46 , wherein the cell is attached to the microplate. 
     
     
         67 . The method of  claim 46 , wherein the device comprises at least one structure configured to enclose a region and control temperature within the region. 
     
     
         68 . The method of  claim 67 , wherein the device is configured to perform temperature cycling of the microplate. 
     
     
         69 . The method of  claim 46 , wherein the device comprises at least one manifold coupled to the structure, wherein the at least one manifold comprises at least one of a reagent aspiration manifold and a reagent dispense manifold. 
     
     
         70 . The method of  claim 46 , wherein the device comprises an integrated system of one or more interconnected chambers, ports, and channels in fluid communication and configured for carrying out an analytical reaction or process, either alone or in cooperation with an appliance or instrument that provides support functions. 
     
     
         71 . The method of  claim 46 , wherein the microplate comprises 4, 6, 12, 24, 48, 96, 384 or 1536 sample wells.