System and method for automated single cell processing and analyses

Abstract

A system and method for automated single cell capture and processing is described, where the system includes a deck supporting and positioning a set of sample processing elements (including an integrated imaging subsystem); a gantry for actuating tools for interactions with the set of sample processing elements supported by the deck; and a base supporting various processing subsystems and a control subsystems in communication with the processing subsystems. The system can automatically execute workflows associated with single cell processing, including antibody detection, other protein detection, mRNA detection, and/or other applications associated with spatial transcriptomics.

What is claimed is: 
     
         1 . A method for detecting a set of targets of a sample, the method comprising:
 capturing a set of cells of the sample in proximity to a set of functionalized particles, at a sample processing substrate;   performing lysis of the set of cells, at the sample processing substrate, thereby allowing binding of the set of targets from the set of cells to the set of functionalized particles;   performing a reverse transcription operation, at the sample processing substrate, with the set of targets bound to oligonucleotides of the set of functionalized particles;   with products of the reverse transcription operation, performing a second strand synthesis operation with the set of functionalized particles;   promoting hybridization of a first probe to a first subset of targets resulting from the second strand synthesis operation;   optically detecting the first probe at the sample processing substrate, thereby characterizing presence of the first subset of targets of the sample;   dehybridizing the first probe; and   promoting hybridization of a second probe to a second subset of targets resulting from the second strand synthesis operation.   
     
     
         2 . The method of  claim 1 , further comprising: optically detecting the second probe at the sample processing substrate, thereby characterizing presence of the second subset of targets of the sample. 
     
     
         3 . The method of  claim 2 , further comprising: dehybridizing the second probe. 
     
     
         4 . The method of  claim 2 , further comprising generating an image dataset, from optical detection of the first probe and the second probe, thereby spatially characterizing distributions of the first subset of targets and the second subset of targets of the sample. 
     
     
         5 . The method of  claim 1 , further comprising generating an image dataset, from optical detection of the first probe, thereby spatially characterizing distributions of the first subset of targets of the sample. 
     
     
         6 . The method of  claim 1 , wherein each of the set of functionalized particles is functionalized with an oligonucleotide comprising a barcode and a capture segment for messenger RNA capture. 
     
     
         7 . The method of  claim 1 , wherein each of the set of functionalized particles is functionalized with an oligonucleotide comprising a barcode and a gene-specific segment corresponding to a target gene of the set of targets. 
     
     
         8 . The method of  claim 1 , wherein the set of targets comprises a panel of mRNA molecules, the method further comprising performing a spatial transcriptomics analysis of the panel of mRNA molecules, with the sample distributed across the sample processing substrate. 
     
     
         9 . The method of  claim 1 , wherein the set of targets comprises a panel of antibodies for assessment of immune responses from a subject, the panel of antibodies comprising one or more of: IgM antibodies, IgG antibodies, IgD antibodies, IgA antibodies, and IgE antibodies. 
     
     
         10 . The method of  claim 1 , wherein performing a second strand synthesis operation is omitted, and wherein the first probe hybridizes to the first subset of targets, wherein the first subset of targets comprises cDNA. 
     
     
         11 . The method of  claim 1 , wherein performing a reverse transcription operation is omitted, wherein the first probe hybridizes to the first subset of targets, and wherein the first subset of target comprises RNA. 
     
     
         12 . A method for detecting a set of targets derived from a set of single cells of a sample, the method comprising:
 at a sample processing substrate comprising a set of features, binding the set of targets derived from cellular lysate to the set of features;   performing a reverse transcription operation, at the sample processing substrate, with the set of targets bound to the set of features;   with products of the reverse transcription operation, performing a second strand synthesis operation with the set of features;   hybridizing a first probe to a first subset of targets resulting from the second strand synthesis operation;   detecting the first probe, thereby characterizing presence of the first subset of targets of the sample at single-cell resolution;   dehybridizing the first probe;   promoting hybridization of a second probe to a second subset of targets resulting from the second strand synthesis operation; and   detecting the second first probe, thereby characterizing presence of the second subset of targets of the sample at single-cell resolution.   
     
     
         13 . The method of  claim 12 , further comprising generating an image dataset, from optical detection of the first probe and the second probe, thereby spatially characterizing distributions of the first subset of targets and the second subset of targets of the sample. 
     
     
         14 . The method of  claim 12 , further comprising generating a single-cell phenotypic analysis from detection of the first probe and the second probe, and performing single cell transcriptomic library preparation from the sample. 
     
     
         15 . The method of  claim 12 , wherein each of the set of features is functionalized with an oligonucleotide comprising a barcode, a unique molecule identifier (UMI), and a capture segment for messenger RNA capture. 
     
     
         16 . The method of  claim 12 , wherein the set of targets comprises a panel of mRNA molecules, the method further comprising performing a spatial transcriptomics analysis of the panel of mRNA molecules, with the sample distributed across the sample processing substrate. 
     
     
         17 . A method for detecting a set of targets of a tissue sample, the method comprising:
 aligning the tissue sample with a first set of features of a substrate, the first set of features functionalized for target capture;   binding the set of targets from the tissue sample to the first set of features;   performing a reverse transcription operation, at the substrate, with the set of targets;   with products of the reverse transcription operation, performing a second strand synthesis operation;   hybridizing a set of probes to outputs of the second strand synthesis operation, the set of probes comprising: a first probe corresponding to a first subset of targets and a second probe corresponding to a second subset of targets;   detecting the first probe, thereby characterizing presence of the first subset of targets of the tissue sample; and   detecting the second first probe, thereby characterizing presence of the second subset of targets of the tissue sample.   
     
     
         18 . The method of  claim 17 , wherein aligning the tissue sample comprises positioning a manifold containing the substrate into alignment with the tissue sample mounted at a slide, and promoting flow of the set of targets from the tissue sample to the first set of features. 
     
     
         19 . The method of  claim 17 , wherein hybridizing the set of probes comprises hybridizing the first probe, comprising a first fluorophore; de-hybridizing and washing the first probe; and hybridizing the second probe, comprising a second fluorophore. 
     
     
         20 . The method of  claim 17 , wherein the first set of features comprises from 1,000 to 10,000,000 features. 
     
     
         21 . The method of  claim 17 , further comprising performing an mRNA quantitation operation with estimation of initial concentrations of mRNAs captured, based upon a set of fluorescence values generated from detection of the first probe and the second probe. 
     
     
         22 . The method of  claim 17 , wherein the set of probes has a probe number (P), wherein the set of probes comprises a set of fluorophores having a fluorophore number (F), each having a set of states (M), thereby enabling multiplexing of M*F P  targets of the tissue sample.