Multimodal readouts for quantifying and sequencing nucleic acids in single cells

Abstract

Provided herein are methods for generating single-cell molecular analysis comprising a) delivering one or more proximity dependent probes to a cell population, wherein each proximity dependent probe comprises a target binding region configured to bind a target RNA and a primer binding site region; b) linking bound proximity dependent probes; c) isolating single cells from the cell population in separate individual discrete volumes, the individual discrete volumes further comprising a primer pair and amplification reagents, wherein the primer pair binds to the primer binding sites of the ligation dependent probes, and wherein at least one primer comprises a barcode sequence that uniquely identifies the individual discrete volume; d) amplifying the ligated probes using the primer pair, wherein the barcode is incorporated into each resulting amplicon; and e) quantifying target RNAs in each individual cell based at least in part on sequencing the resulting amplicons.

1 .- 123 . (canceled) 
     
     
         124 . A method for generating single-cell molecular analysis comprising;
 a) delivering one or more proximity dependent probes to a cell population, wherein each proximity dependent probe comprises a target binding region configured to bind one or more target RNAs and a primer binding site region;   b) linking bound proximity dependent probes;   c) using combinatorial split-and-pool strategies, optionally ligation, to add sequential barcodes to the linked proximity dependent probes to attach a unique barcode to the set of probes derived from single cells;   d) amplifying the ligated probes using the primer pair, wherein the barcode is incorporated into each resulting amplicon; and   e) quantifying target RNAs in each individual cell based at least in part on sequencing the resulting amplicons.   
     
     
         125 . The method of  claim 124 , further comprising delivering DNA-tagged protein binding molecules, amplifying the DNA tags to generate sequencing amplicons and quantifying target protein abundance based at least in part on sequencing of amplicons. 
     
     
         126 . The method of  claim 125 , wherein the protein binding molecule is an aptamer or an antibody. 
     
     
         127 . The method of  claim 124 , wherein the bound proximity dependent probes are linked by ligation, splinted ligation, hybridization, or proximity extension. 
     
     
         128 . The method of  claim 124 , wherein the proximity dependent probes are molecular inversion probes (MIPs), HyPR probes, padlock probes, or split-ligation probes, each probe further comprising a unique molecular identifier (UMI). 
     
     
         129 . The method of  claim 124 , wherein the one or more proximity dependent probes target at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 500, at least 1000, or at least 10,000 target RNAs. 
     
     
         130 . The method of  claim 129 , wherein multiple proximity dependent probes bind to the same target RNA. 
     
     
         131 . The method of  claim 130 , wherein 2 to 100 proximity dependent probes are used per target RNA. 
     
     
         132 . A molecular assay system comprising;
 a) a set of proximity dependent probes; and   b) a set of primer pairs, wherein each primer pair comprises at least one barcoded primer.   
     
     
         133 . The system of  claim 132 , wherein the proximity dependent probes are molecular inversion probes (MIPs), HyPR probes, padlock probes, or split-ligation probes, each probe further comprising a unique molecular identifier (UMI). 
     
     
         134 . The system of  claim 132 , wherein the set of proximity dependent probes comprise proximity dependent probes for detecting and/or quantitating at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 500, at least 1000, or at least 10,000 target RNAs. 
     
     
         135 . The system of  claim 132 , wherein 2 to 100 proximity dependent probes are used per target RNA. 
     
     
         136 . The system of  claim 132 , wherein the set of proximity dependent probes detect gene expression markers on one or more cell signaling pathways. 
     
     
         137 . The system of  claim 136 , wherein the one or more cell signaling pathways comprises a cell development pathway, a cancer signaling pathway, or an immune response signaling pathway. 
     
     
         138 . The system of  claim 132 , wherein the primer pairs amplify one or more genomic DNA loci and allow for genotyping in combination with targeted RNA detection and quantitation. 
     
     
         139 . The system of  claim 138 , wherein the targeted genomic DNA loci include sites of somatic mutations that affect known processes such as proliferation or cancer development. 
     
     
         140 . The system of  claim 132 , wherein the individual discrete volumes are droplets, wherein the kit further comprises reagents for droplet formation. 
     
     
         141 . The system of  claim 140 , further comprising a means for sorting and or encapsulating individual cells into droplets. 
     
     
         142 . The system of  claim 141 , wherein the means for sorting and/or encapsulating individual cells comprises a microfluidic device. 
     
     
         143 . The system of  claim 132 , further comprising reagents for PCR amplification. 
     
     
         144 . The system of  claim 132 , wherein one or both barcoded primers comprise a set of discrete beads, wherein each bead contains a unique barcode. 
     
     
         145 . The system of  claim 144 , wherein the discrete bead comprises hydrogel beads, magnetic beads, or other beads. 
     
     
         146 . The system of  claim 144 , wherein the discrete beads are distributed randomly amongst the droplets together with cells so that each droplet has ˜1 cell and ˜1 bead with barcodes. 
     
     
         147 . A molecular assay system comprising droplet forming reagents for formation of hydrogel based droplets that contain cells and/or primers with linkers that link the primer pairs to the hydrogel matrix upon droplet formation. 
     
     
         148 . The system of  claim 147 , wherein the droplet forming reagents further comprise a linker molecule for linking nucleic acids to the hydrogel and/or a linker molecule for linking proteins to the hydrogel and/or primers with releasable linkers that link amplification primers to the hydrogel matrix upon droplet formation. 
     
     
         149 . The system of  claim 148 , wherein the nucleic acid linking molecule is LabelX and the protein linking molecule is AcX. 
     
     
         150 . The system of  claim 147 , further comprising oligo-dT RT primers comprising a releasable linker that links the oligo-dT RT primers to the hydrogel. 
     
     
         151 . The system of  claim 147 , further comprising a set of proximity dependent probes. 
     
     
         152 . The system of  claim 151 , wherein the proximity dependent probes are molecular inversion probes (MIPs), HyPR probes, padlock probes, or split-ligation probes, each probe further comprising a unique molecular identifier (UMI). 
     
     
         153 . The system of  claim 152 , wherein the set of proximity dependent probes comprise proximity dependent probes for detecting and/or quantitating at least 1, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 500, at least 1000, or at least 10,000 target RNAs or DNAs. 
     
     
         154 . The system of  claim 153 , wherein 10 to 100 proximity dependent probes are used per target RNA and/or DNA. 
     
     
         155 . The system of  claim 147 , further comprising one or more oligonucleotide tagged protein binding molecules, wherein the oligonucleotide on the oligonucleotide tagged protein binding molecules comprises a primer binding set for the primer pairs or a portion of the primer pairs. 
     
     
         156 . The system of  claim 155 , wherein the set of proximity dependent probes and/or oligonucleotide tagged protein binding molecules detect gene expression markers of one or more cell signaling pathways, and optionally wherein the one or more cell signaling pathways comprise a cell development pathway, a cancer signaling pathway, or an immune response signaling pathway. 
     
     
         157 . The system of  claim 147 , further comprising (a) fixing reagents to fix cells prior to encapsulation in hydrogel droplets, (b) cross-linking reversing agents to reverse cross-links formed in fixed cells, (c) combinatorial indexing reagents for adding barcode sequences to the one or more primer pairs linked to the hydrogel matrix, (d) barcoding adapters and reagents for ligating the barcoding adapters to target molecules to allow for direct barcoding of target molecules, (e) an exonuclease for converting dsDNA into ssDNA, (f) whole genome amplification regents, (g) PCR amplification reagents, (h) reverse transcription reagents, (i) rolling circle amplification reagents, or any combination of (a)-(i). 
     
     
         158 . The system of  claim 147 , further comprising a means for sorting, encapsulating individual cells in hydrogel droplets, or both. 
     
     
         159 . The system of  claim 158 , wherein the means for sorting and/or encapsulating individual cells in hydrogel droplets comprises a microfluidic device. 
     
     
         160 . The system of  claim 147 , wherein the droplet reagents comprise acrylamide/bisacrylamide, acrylamide/di-hydroxyethylenebisacrylamide, or acrylamide/N,N′-bis(acryloyl)cystamine. 
     
     
         161 . The system of  claim 160 , wherein the ratio of acrylamide to bisacrylamide ranges from 10:1 to 40:1, wherein the percentage of acrylamide/bisacrylamide ranges from 3% to 20%, or both 
     
     
         162 . The system of  claim 147 , further comprising barcoded beads, wherein the barcoded beads comprise sets of primers are capable of being co-emulsified with the cell-containing hydrogels.