Immobilization-based systems and methods for genetic analysis and other applications

Abstract

The present invention generally relates to microfluidics and labeled nucleic acids. Certain aspects are generally directed to containing cells in gels, such as agarose gels, and determining nucleic acids within the cells, e.g., while contained within the gels. The nucleic acids may be, for example, genomic DNA, mRNA, transcriptomes, or the like. In some embodiments, for instance, both genomic DNA and RNA (e.g., as in a transcriptome) from a cell may be determined. In some cases, the nucleic acids may be attached to beads for sequencing or other purposes. Such systems may be useful, for example, for high-throughput sequencing or other applications.

What is claimed is: 
     
         1 . A method, comprising:
 lysing cells within gel droplets to release DNA and RNA from the cells;   attaching the DNA to a first set of oligonucleotides; and   attaching the RNA to a second set of oligonucleotides.   
     
     
         2 . The method of  claim 1 , wherein the cells are contained within the gel droplets at no more than about 1 cell/gel droplet. 
     
     
         3 . The method of any one of  claim 1  or  2 , wherein the cells are contained within the gel droplets at no more than about 0.1 cell/gel droplet. 
     
     
         4 . The method of any one of  claims 1 - 3 , wherein the cells are contained within the gel droplets at no more than about 0.01 cell/gel droplet. 
     
     
         5 . The method of any one of  claims 1 - 4 , wherein at least about 90% of the gel droplets contains one cell or no cell. 
     
     
         6 . The method of any one of  claims 1 - 5 , wherein at least some of the gel droplets comprise agarose. 
     
     
         7 . The method of any one of  claims 1 - 6 , wherein the agarose has a gelation temperature of less than about 60° C. 
     
     
         8 . The method of any one of  claims 1 - 7 , wherein the agarose has a gelation temperature of less than about 40° C. 
     
     
         9 . The method of any one of  claims 1 - 8 , wherein the gel droplets have an average diameter of no more than about 1 mm. 
     
     
         10 . The method of any one of  claims 1 - 9 , wherein at least some of the gel droplets are contained within a microfluidic channel. 
     
     
         11 . The method of any one of  claims 1 - 10 , comprising lysing at least some of the cells using a cell lysis reagent. 
     
     
         12 . The method of  claim 11 , wherein the cell lysis reagent comprises proteinase K. 
     
     
         13 . The method of any one of  claims 1 - 12 , wherein at least some of the cells are human cells. 
     
     
         14 . The method of any one of  claims 1 - 13 , wherein the DNA includes genomic DNA. 
     
     
         15 . The method of  claim 14 , further comprising fragmenting the genomic DNA after release from the cells. 
     
     
         16 . The method of  claim 15 , wherein fragmenting the genomic DNA comprises exposing the genomic DNA to a restriction endonuclease. 
     
     
         17 . The method of any one of  claims 1 - 16 , wherein the RNA includes mRNA. 
     
     
         18 . The method of  claim 17 , further comprising forming cDNA using the RNA. 
     
     
         19 . The method of  claim 18 , further comprising forming cDNA using the RNA using a reverse transcriptase. 
     
     
         20 . The method of any one of  claim 18  or  19 , further comprising attaching the cDNA to oligonucleotides within the gel droplet. 
     
     
         21 . The method of any one of  claims 1 - 20 , wherein attaching the DNA to the first set of oligonucleotides comprises ligating at least some of the DNA to the oligonucleotides. 
     
     
         22 . The method of any one of  claims 1 - 21 , wherein attaching the RNA to the first set of oligonucleotides comprises ligating at least some of the RNA to the oligonucleotides. 
     
     
         23 . The method of any one of  claims 1 - 22 , wherein first set of oligonucleotides and/or the second set of oligonucleotides are attached to particles. 
     
     
         24 . The method of  claim 23 , wherein the particles comprise hydrogel particles. 
     
     
         25 . The method of any one of  claim 23  or  24 , wherein the particles have an average diameter of no more than about 1 mm. 
     
     
         26 . The method of any one of  claims 23 - 25 , wherein the particles have an average diameter of no more than about 500 micrometers. 
     
     
         27 . The method of any one of  claims 23 - 26 , wherein the particles have an average diameter of no more than about 1 micrometer. 
     
     
         28 . The method of any one of  claims 23 - 27 , wherein the first set of oligonucleotides further comprises a first set of barcode sequences. 
     
     
         29 . The method of any one of  claims 23 - 28 , wherein the second set of oligonucleotides further comprises a second set of barcode sequences. 
     
     
         30 . The method of  claim 29 , wherein the first set of barcode sequences and the second set of barcode sequences have at least one barcode sequence in common. 
     
     
         31 . The method of any one of  claim 29  or  30 , wherein the first set of barcode sequences and/or the second set of barcode sequences comprise a first barcode selected from a pre-defined pool of first barcodes and a second barcode selected from a pre-defined pool of second barcodes, such that substantially each of the particles comprises distinguishable barcode sequences. 
     
     
         32 . The method of  claim 31 , wherein the pre-defined pool of first barcodes comprises at least about 300 distinguishable barcodes. 
     
     
         33 . The method of any one of  claims 31 - 32 , wherein the pre-defined pool of first barcodes comprises at least about 1,000 distinguishable barcodes. 
     
     
         34 . The method of any one of  claims 31 - 33 , wherein the pre-defined pool of second barcodes comprises at least about 300 distinguishable barcodes. 
     
     
         35 . The method of any one of  claims 31 - 34 , wherein the pre-defined pool of second barcodes comprises at least about 1,000 distinguishable barcodes. 
     
     
         36 . The method of any one of  claims 31 - 35 , wherein the pre-defined pool of second barcodes comprises at least about 3,000 distinguishable barcodes. 
     
     
         37 . The method of any one of  claims 31 - 36 , wherein the distinguishable barcode sequences comprises at least 10,000 distinguishable barcode sequences. 
     
     
         38 . The method of any one of  claims 31 - 37 , wherein the distinguishable barcode sequences comprises at least 100,000 distinguishable barcode sequences. 
     
     
         39 . The method of any one of  claims 23 - 38 , wherein at least some of the oligonucleotides are covalently bonded to the particles. 
     
     
         40 . The method of  claim 39 , wherein at least some oligonucleotides of the first set of oligonucleotides and/or the second set of oligonucleotides are covalently bonded to the particles via an acrylic phosphoramidite linkage. 
     
     
         41 . The method of any one of  claim 39  or  40 , wherein at least some oligonucleotides of the first set of oligonucleotides and/or the second set of oligonucleotides are covalently bonded to the particles via an amino linkage. 
     
     
         42 . The method of any one of  claims 23 - 41 , wherein at least some oligonucleotides of the first set of oligonucleotides and/or the second set of oligonucleotides are attached to the particles via a biotin-steptavidin linkage. 
     
     
         43 . The method of any one of  claims 23 - 42 , wherein at least some oligonucleotides of the first set of oligonucleotides and/or the second set of oligonucleotides comprise a cleavable linker. 
     
     
         44 . The method of  claim 43 , wherein the cleavable linker is a photocleavable linker. 
     
     
         45 . The method of  claim 43 , wherein the cleavable linker is a chemically cleavable linker. 
     
     
         46 . The method of  claim 43 , wherein the cleavable linker is an enzymatically cleavable linker. 
     
     
         47 . The method of any one of  claims 23 - 46 , further comprising amplifying at least some oligonucleotides of the first set of oligonucleotides and/or the second set of oligonucleotides. 
     
     
         48 . The method of  claim 47 , comprising amplifying the oligonucleotides using PCR. 
     
     
         49 . The method of any one of  claims 1 - 48 , further comprising encapsulating the gel droplet within a fluidic droplet prior to amplifying the oligonucleotides. 
     
     
         50 . The method of  claim 49 , wherein the fluidic droplets contain the particles. 
     
     
         51 . The method of  claim 50 , wherein the particles are contained within the fluidic droplets at no more than about 1 particle/droplet. 
     
     
         52 . The method of any one of  claim 50  or  51 , wherein the particles are contained within the fluidic droplets at no more than about 0.1 particle/droplet. 
     
     
         53 . The method of any one of  claims 50 - 52 , wherein the particles are contained within the fluidic droplets at no more than about 0.01 particle/droplet. 
     
     
         54 . The method of any one of  claims 50 - 53 , wherein at least about 90% of the fluidic droplets contains one particle or no particle. 
     
     
         55 . The method of any one of  claims 47 - 54 , further comprising releasing at least some of the oligonucleotides of the first set of oligonucleotides and/or the second set of oligonucleotides from the particles. 
     
     
         56 . The method of  claim 55 , comprising cleaving at least some of the oligonucleotides from the particles. 
     
     
         57 . The method of any one of  claim 55  or  56 , comprising photocleaving at least some of the oligonucleotides from the particles. 
     
     
         58 . The method of any one of  claims 55 - 57 , comprising releasing the oligonucleotides from the particles prior to amplifying the oligonucleotides. 
     
     
         59 . The method of any one of  claims 47 - 58 , further comprising bursting the fluidic droplet after amplifying the oligonucleotides. 
     
     
         60 . The method of  claim 59 , further comprising sequencing at least some of the first set of oligonucleotides and/or the second set of oligonucleotides. 
     
     
         61 . A method, comprising:
 lysing a cell within a gel droplet to release RNA from the cell;   forming cDNA using the RNA; and   attaching the cDNA to an oligonucleotide within the gel droplet.   
     
     
         62 . The method of  claim 61 , comprising attaching the cDNA to the oligonucleotides using a ligase. 
     
     
         63 . The method of any one of  claim 61  or  62 , comprising exposing the RNA to a linear polyacrylamide. 
     
     
         64 . The method of any one of  claims 61 - 63 , comprising forming cDNA using the RNA by exposing the RNA to a reverse transcriptase. 
     
     
         65 . The method of  claim 64 , wherein the reverse transcriptase adds three unpaired A bases to the cDNA. 
     
     
         66 . The method of  claim 65 , wherein attaching the cDNA to the oligonucleotide comprises matching the three unpaired A bases to the cDNA to an oligonucleotide comprising three unpaired T bases. 
     
     
         67 . The method of any one of  claims 61 - 66 , wherein the cells are contained within the gel droplets at no more than about 1 cell/gel droplet. 
     
     
         68 . The method of any one of  claims 61 - 67 , wherein the cells are contained within the gel droplets at no more than about 0.1 cell/gel droplet. 
     
     
         69 . The method of any one of  claims 61 - 68 , wherein the cells are contained within the gel droplets at no more than about 0.01 cell/gel droplet. 
     
     
         70 . The method of any one of  claims 61 - 69 , wherein at least about 90% of the gel droplets contains one cell or no cell. 
     
     
         71 . The method of any one of  claims 61 - 70 , wherein at least some of the gel droplets comprise agarose. 
     
     
         72 . The method of any one of  claims 61 - 71 , wherein the agarose has a gelation temperature of less than about 60° C. 
     
     
         73 . The method of any one of  claims 61 - 72 , wherein the agarose has a gelation temperature of less than about 40° C. 
     
     
         74 . The method of any one of  claims 61 - 73 , wherein the gel droplets have an average diameter of no more than about 1 mm. 
     
     
         75 . The method of any one of  claims 61 - 74 , comprising lysing at least some of the cells using a cell lysis reagent. 
     
     
         76 . The method of  claim 75 , wherein the cell lysis reagent comprises proteinase K. 
     
     
         77 . The method of any one of  claims 61 - 76 , wherein at least some of the cells are human cells. 
     
     
         78 . The method of any one of  claims 61 - 77 , wherein the RNA includes miRNA. 
     
     
         79 . The method of any one of  claims 61 - 78 , wherein the RNA includes mRNA. 
     
     
         80 . The method of any one of  claims 61 - 79 , wherein at least some of the oligonucleotides are attached to particles. 
     
     
         81 . The method of  claim 80 , wherein the particles comprise hydrogel particles. 
     
     
         82 . The method of any one of  claim 80  or  81 , wherein the particles have an average diameter of no more than about 1 mm. 
     
     
         83 . The method of any one of  claims 80 - 82 , wherein the particles have an average diameter of no more than about 500 micrometers. 
     
     
         84 . The method of any one of  claims 80 - 83 , wherein the particles have an average diameter of no more than about 1 micrometer. 
     
     
         85 . The method of any one of  claims 80 - 84 , wherein at least some of the oligonucleotides are covalently bonded to the particles. 
     
     
         86 . The method of  claim 85 , wherein at least some oligonucleotides are covalently bonded to the particles via an acrylic phosphoramidite linkage. 
     
     
         87 . The method of any one of  claim 85  or  86 , wherein at least some oligonucleotides are covalently bonded to the particles via an amino linkage. 
     
     
         88 . The method of any one of  claims 80 - 87 , wherein the oligonucleotides are attached to the particles via a biotin-steptavidin linkage. 
     
     
         89 . The method of any one of  claims 80 - 88 , wherein the oligonucleotides comprise a cleavable linker. 
     
     
         90 . The method of  claim 89 , wherein the cleavable linker is a photocleavable linker. 
     
     
         91 . The method of  claim 89 , wherein the cleavable linker is a chemically cleavable linker. 
     
     
         92 . The method of  claim 89 , wherein the cleavable linker is an enzymatically cleavable linker. 
     
     
         93 . The method of any one of  claims 80 - 92 , wherein the oligonucleotides further comprises a barcode sequence. 
     
     
         94 . The method of  claim 93 , wherein the barcode sequences comprise a first barcode selected from a pre-defined pool of first barcodes and a second barcode selected from a pre-defined pool of second barcodes, such that substantially each of the particles comprises distinguishable barcode sequences. 
     
     
         95 . The method of any one of  claim 93  or  94 , wherein the pre-defined pool of first barcodes comprises at least about 300 distinguishable barcodes. 
     
     
         96 . The method of  claim 95 , wherein the pre-defined pool of first barcodes comprises at least about 1,000 distinguishable barcodes. 
     
     
         97 . The method of any one of  claim 95  or  96 , wherein the pre-defined pool of second barcodes comprises at least about 300 distinguishable barcodes. 
     
     
         98 . The method of any one of  claims 95 - 97 , wherein the pre-defined pool of second barcodes comprises at least about 1,000 distinguishable barcodes. 
     
     
         99 . The method of any one of  claims 95 - 98 , wherein the pre-defined pool of second barcodes comprises at least about 3,000 distinguishable barcodes. 
     
     
         100 . The method of any one of  claims 95 - 99 , wherein the distinguishable barcode sequences comprises at least 10,000 distinguishable barcode sequences. 
     
     
         101 . The method of any one of  claims 95 - 100 , wherein the distinguishable barcode sequences comprises at least 100,000 distinguishable barcode sequences. 
     
     
         102 . The method of any one of  claims 80 - 101 , further comprising amplifying at least some of the oligonucleotides. 
     
     
         103 . The method of  claim 102 , comprising amplifying the oligonucleotides using PCR. 
     
     
         104 . The method of any one of  claims 61 - 103 , further comprising encapsulating the gel droplet within a fluidic droplet prior to amplifying the oligonucleotides. 
     
     
         105 . The method of  claim 104 , wherein the fluidic droplets contain the particles. 
     
     
         106 . The method of  claim 105 , wherein the particles are contained within the fluidic droplets at no more than about 1 particle/droplet. 
     
     
         107 . The method of any one of  claim 105  or  106 , wherein the particles are contained within the fluidic droplets at no more than about 0.1 particle/droplet. 
     
     
         108 . The method of any one of  claims 105 - 107 , wherein the particles are contained within the fluidic droplets at no more than about 0.01 particle/droplet. 
     
     
         109 . The method of any one of  claims 105 - 108 , wherein at least about 90% of the fluidic droplets contains one particle or no particle. 
     
     
         110 . The method of any one of  claims 105 - 109 , further comprising releasing at least some of the oligonucleotides from the particles. 
     
     
         111 . The method of  claim 110 , comprising cleaving at least some of the oligonucleotides from the particles. 
     
     
         112 . The method of any one of  claim 110  or  111 , comprising photocleaving at least some of the oligonucleotides from the particles. 
     
     
         113 . The method of any one of  claims 110 - 112 , comprising releasing the oligonucleotides from the particles prior to amplifying the oligonucleotides. 
     
     
         114 . The method of  claim 113 , further comprising bursting the fluidic droplet after amplifying the oligonucleotides. 
     
     
         115 . The method of any one of  claim 113  or  114 , further comprising sequencing at least some of the oligonucleotides. 
     
     
         116 . An article, comprising:
 a gel droplet comprising a lysed cell, DNA attached to a first set of oligonucleotides, and RNA attached to a second set of oligonucleotides.   
     
     
         117 . The composition of  claim 116 , wherein the cells are contained within the gel droplets at no more than about 1 cell/gel droplet. 
     
     
         118 . The composition of any one of  claim 116  or  117 , wherein the cells are contained within the gel droplets at no more than about 0.1 cell/gel droplet. 
     
     
         119 . The composition of any one of  claims 116 - 118 , wherein the cells are contained within the gel droplets at no more than about 0.01 cell/gel droplet. 
     
     
         120 . The composition of any one of  claims 116 - 119 , wherein at least about 90% of the gel droplets contains one cell or no cell. 
     
     
         121 . The composition of any one of  claims 116 - 120 , wherein at least some of the gel droplets comprise agarose. 
     
     
         122 . The composition of any one of  claims 116 - 121 , wherein the agarose has a gelation temperature of less than about 60° C. 
     
     
         123 . The composition of any one of  claims 116 - 122 , wherein the agarose has a gelation temperature of less than about 40° C. 
     
     
         124 . The composition of any one of  claims 116 - 123 , wherein the gel droplets have an average diameter of no more than about 1 mm. 
     
     
         125 . The composition of any one of  claims 116 - 124 , wherein at least some of the gel droplets are contained within a microfluidic channel. 
     
     
         126 . The composition of any one of  claims 116 - 125 , wherein at least some of the cells are human cells. 
     
     
         127 . The composition of any one of  claims 116 - 126 , wherein the DNA includes genomic DNA. 
     
     
         128 . The composition of any one of  claims 116 - 127 , wherein the RNA includes mRNA. 
     
     
         129 . The composition of any one of  claims 116 - 128 , wherein first set of oligonucleotides and/or the second set of oligonucleotides are attached to particles. 
     
     
         130 . The composition of  claim 129 , wherein the particles comprise hydrogel particles. 
     
     
         131 . The composition of any one of  claim 129  or  130 , wherein the particles have an average diameter of no more than about 1 mm. 
     
     
         132 . The composition of any one of  claims 129 - 131 , wherein the particles have an average diameter of no more than about 500 micrometers. 
     
     
         133 . The composition of any one of  claims 129 - 132 , wherein the particles have an average diameter of no more than about 1 micrometer. 
     
     
         134 . The composition of any one of  claims 129 - 133 , wherein the first set of oligonucleotides further comprises a first set of barcode sequences. 
     
     
         135 . The composition of any one of  claims 129 - 134 , wherein the second set of oligonucleotides further comprises a second set of barcode sequences. 
     
     
         136 . The composition of  claim 135 , wherein the first set of barcode sequences and the second set of barcode sequences have at least one barcode sequence in common. 
     
     
         137 . The composition of any one of  claims 135  or  136 , wherein the first set of barcode sequences and/or the second set of barcode sequences comprise a first barcode selected from a pre-defined pool of first barcodes and a second barcode selected from a pre-defined pool of second barcodes, such that substantially each of the particles comprises distinguishable barcode sequences. 
     
     
         138 . The composition of  claim 137 , wherein the pre-defined pool of first barcodes comprises at least about 300 distinguishable barcodes. 
     
     
         139 . The composition of any one of  claim 137  or  138 , wherein the pre-defined pool of first barcodes comprises at least about 1,000 distinguishable barcodes. 
     
     
         140 . The composition of any one of  claims 137 - 139 , wherein the pre-defined pool of second barcodes comprises at least about 300 distinguishable barcodes. 
     
     
         141 . The composition of any one of  claims 137 - 140 , wherein the pre-defined pool of second barcodes comprises at least about 1,000 distinguishable barcodes. 
     
     
         142 . The composition of any one of  claims 137 - 141 , wherein the pre-defined pool of second barcodes comprises at least about 3,000 distinguishable barcodes. 
     
     
         143 . The composition of any one of  claims 137 - 142 , wherein the distinguishable barcode sequences comprises at least 10,000 distinguishable barcode sequences. 
     
     
         144 . The composition of any one of  claims 137 - 143 , wherein the distinguishable barcode sequences comprises at least 100,000 distinguishable barcode sequences. 
     
     
         145 . The composition of any one of  claims 129 - 144 , wherein at least some of the oligonucleotides are covalently bonded to the particles. 
     
     
         146 . The composition of  claim 145 , wherein at least some oligonucleotides of the first set of oligonucleotides and/or the second set of oligonucleotides are covalently bonded to the particles via an acrylic phosphoramidite linkage. 
     
     
         147 . The composition of any one of  claim 145  or  146 , wherein at least some oligonucleotides of the first set of oligonucleotides and/or the second set of oligonucleotides are covalently bonded to the particles via an amino linkage. 
     
     
         148 . The composition of any one of  claims 129 - 147 , wherein at least some oligonucleotides of the first set of oligonucleotides and/or the second set of oligonucleotides are attached to the particles via a biotin-steptavidin linkage. 
     
     
         149 . The composition of any one of  claims 129 - 148 , wherein at least some oligonucleotides of the first set of oligonucleotides and/or the second set of oligonucleotides comprise a cleavable linker. 
     
     
         150 . The composition of  claim 149 , wherein the cleavable linker is a photocleavable linker. 
     
     
         151 . The composition of  claim 149 , wherein the cleavable linker is a chemically cleavable linker. 
     
     
         152 . The composition of  claim 149 , wherein the cleavable linker is an enzymatically cleavable linker. 
     
     
         153 . The composition of any one of  claims 116 - 152 , wherein the gel droplet is encapsulated within a fluidic droplet. 
     
     
         154 . The composition of  claim 153 , wherein the fluidic droplets contain the particles. 
     
     
         155 . The composition of  claim 154 , wherein the particles are contained within the fluidic droplets at no more than about 1 particle/droplet. 
     
     
         156 . The composition of any one of  claim 154  or  155 , wherein the particles are contained within the fluidic droplets at no more than about 0.1 particle/droplet. 
     
     
         157 . The composition of any one of  claims 154 - 156 , wherein the particles are contained within the fluidic droplets at no more than about 0.01 particle/droplet. 
     
     
         158 . The composition of any one of  claims 154 - 157 , wherein at least about 90% of the fluidic droplets contains one particle or no particle.