Spatially addressable molecular barcoding

Abstract

The disclosure provides for methods, compositions, systems, devices, and kits for determining the number of distinct targets in distinct spatial locations within a sample. In some examples, the methods include: stochastically barcoding the plurality of targets in the sample using a plurality of stochastic barcodes, wherein each of the plurality of stochastic barcodes comprises a spatial label and a molecular label; estimating the number of each of the plurality of targets using the molecular label; and identifying the spatial location of each of the plurality of targets using the spatial label. The method can be multiplexed.

What is claimed is: 
     
       1. A method for determining spatial locations of a plurality of single cells, comprising:
 stochastically barcoding the plurality of single cells using a plurality of synthetic particles,
 wherein each of the plurality of synthetic particles comprises a plurality of stochastic barcodes, a first group of optical labels, and a second group of optical labels, 
 wherein each of the plurality of stochastic barcodes comprises a cellular label and a molecular label, 
 wherein each optical label in the first group of optical labels comprises a first optical moiety and each optical label in the second group of optical labels comprises a second optical moiety, and 
 wherein each of the plurality of synthetic particles is associated with an optical barcode comprising the first optical moiety and the second optical moiety; 
 
 detecting the optical barcode of each of the plurality of synthetic particles to determine the location of each of the plurality of synthetic particles; and 
 determining the spatial locations of the plurality of single cells based on the locations of the plurality of synthetic particles. 
 
     
     
       2. The method of  claim 1 , wherein the first optical moiety and the second optical moiety are selected from a group comprising consisting of two or more spectrally-distinct optical moieties. 
     
     
       3. The method of  claim 1 , wherein stochastically barcoding the plurality of single cells using the plurality of synthetic particles comprises contacting the plurality of single cells with the plurality of synthetic particles. 
     
     
       4. The method of  claim 3 , wherein a synthetic particle of the plurality of synthetic particles is in close proximity to a single cell or a small number of cells. 
     
     
       5. The method of  claim 3 ,
 wherein each of the plurality of single cells comprises a plurality of targets, 
 wherein stochastically barcoding the plurality of single cells further comprises hybridizing the plurality of stochastic barcodes with the plurality of targets to generate stochastically barcoded targets, and 
 wherein at least one of the plurality of targets is hybridized to one of the plurality of stochastic barcodes. 
 
     
     
       6. The method of  claim 1 , wherein cellular labels of at least two stochastic barcodes of the plurality of stochastic barcodes on one synthetic particle have the same sequence, and wherein cellular labels of at least two stochastic barcodes of the plurality of stochastic barcodes on different synthetic particles have different sequences. 
     
     
       7. The method of  claim 1 , wherein molecular labels of at least two stochastic barcodes of the plurality of stochastic barcodes on one synthetic particle have different sequences. 
     
     
       8. The method of  claim 1 , wherein the molecular labels are selected from a group comprising consisting of at least 100 molecular labels with unique sequences. 
     
     
       9. The method of  claim 1 , wherein the molecular labels are selected from a group comprising consisting of at least 1000 molecular labels with unique sequences. 
     
     
       10. The method of  claim 1 , wherein detecting the optical barcode of each of the plurality of synthetic particles to determine the location of each of the plurality of synthetic particles comprises generating an optical image showing the optical barcodes and the locations of the plurality of synthetic particles. 
     
     
       11. The method of  claim 1 , wherein the plurality of single cells comprises cells distributed across a microwell array comprising microwells. 
     
     
       12. The method of  claim 11 , wherein each of the plurality of single cells comprises a plurality of targets, the method comprising:
 lysing the plurality of single cells; and 
 generating an indexed library of stochastically barcoded targets,
 wherein generating an indexed library of stochastically barcoded targets comprises hybridizing the plurality of stochastic barcodes with the plurality of targets to generate stochastically barcoded targets, 
 wherein the molecular label comprises a molecular label sequence, 
 wherein the cellular label comprises a cellular label sequence, and  
 wherein each of the stochastically barcoded targets comprises a cellular label sequence, a molecular label sequence, and at least a portion of the a complementary sequence of one of the plurality of targets. 
 
 
     
     
       13. The method of  claim 12 , comprising:
 amplifying the stochastically barcoded targets of the indexed library to generate amplified stochastically barcoded targets; and 
 sequencing the amplified stochastically barcoded targets to determine the number of amplified stochastically barcoded targets with unique molecular label sequences and identical complementary sequence,
 wherein the number of amplified stochastically barcoded targets with unique molecular label sequences and identical complementary sequence is substantially the same as the occurrences of targets with sequences complementary of the identical complementary sequence in the single cell or the small number of cells. 
 
 
     
     
       14. The method of  claim 13 , wherein the labeled target molecules are amplified using bridge amplification, amplification with a gene specific primer, amplification with a universal primer, amplification with an oligo(dT) primer, or any combination thereof. 
     
     
       15. The method of  claim 1 , wherein the plurality of single cells comprises a tissue, a cell monolayer, fixed cells, a tissue section, or any combination thereof. 
     
     
       16. The method of  claim 1 , wherein a synthetic particle of the plurality of synthetic particle particles is a bead. 
     
     
       17. The method of  claim 16 , wherein the bead is selected from the group comprising consisting of streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A/G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbead, anti-fluorochrome microbead, and any combination thereof. 
     
     
       18. The method of  claim 1 , wherein a synthetic particle of the plurality of synthetic particles comprises a material selected from the group comprising consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof. 
     
     
       19. A synthetic particle composition, comprising:
 a synthetic particle;  
 a plurality of stochastic barcodes, wherein each of the plurality of stochastic barcodes comprises a cellular label and a molecular label; 
 a first group of optical labels; and 
 a second group of optical labels,
 wherein the plurality of stochastic barcodes, the first group of optical labels, and the second group of optical labels are attached to the surface of the synthetic particle,  
 wherein each optical label in the first group of optical labels comprises a first optical moiety and each optical label in the second group of optical labels comprises a second optical moiety, and 
 wherein each of the plurality of synthetic particles the synthetic particle is associated with an optical barcode comprising the first optical moiety and the second optical moiety. 
 
 
     
     
       20. The synthetic particle composition of  claim 19 , wherein the molecular labels of the plurality of stochastic barcodes are different from one another, and the molecular labels are selected from a group comprising consisting of at least 100 molecular labels with unique sequences. 
     
     
       21. The synthetic particle composition of  claim 19 , wherein cellular labels of at least two stochastic barcodes of the plurality of stochastic barcodes have the same sequence. 
     
     
       22. The synthetic particle composition of  claim 19 , wherein molecular labels of at least two stochastic barcodes of the plurality of stochastic barcodes have different sequences. 
     
     
       23. The synthetic particle composition of  claim 19 , wherein molecular labels of the plurality of stochastic barcodes are selected from a group comprising consisting of at least 100 molecular labels with unique sequences. 
     
     
       24. The synthetic particle composition of  claim 19 , wherein molecular labels of the plurality of stochastic barcodes are selected from a group comprising consisting of at least 1000 molecular labels with unique sequences. 
     
     
       25. The synthetic particle composition of  claim 19 , wherein the first optical moiety and the second optical moiety are selected from a group comprising consisting two or more spectrally-distinct optical moieties. 
     
     
       26. The synthetic particle composition of  claim 19 ,
 wherein each of the plurality of stochastic barcodes comprises a spatial label, and 
 wherein spatial labels of at least two stochastic barcodes of the plurality of stochastic barcodes differ from each other by at least one nucleotide. 
 
     
     
       27. The synthetic particle composition of  claim 19 ,
 wherein each of the plurality of stochastic barcodes further comprises a universal label, and 
 wherein universal labels of at least two stochastic barcodes of the plurality of stochastic barcodes have the same sequence. 
 
     
     
       28. The synthetic particle composition of  claim 19 , wherein the synthetic particle is a bead. 
     
     
       29. The synthetic particle composition of  claim 28 , wherein the bead is selected from the group comprising consisting of streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A/G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbead, anti-fluorochrome microbead, and any combination thereof. 
     
     
       30. The synthetic particle composition of  claim 19 , wherein the synthetic particle comprises a material selected from the group comprising consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof. 
     
     
       31. A method for determining the number and spatial locations of a plurality of targets in a sample, comprising:
 providing a solid support comprising a plurality of synthetic particles associated with a plurality of stochastic barcodes, wherein each of the plurality of stochastic barcodes comprises a spatial label and a molecular label;   decoding the solid support by contacting the solid support with a plurality of decoding nucleic acids labeled with a decoding label and detecting the presence of the decoding label,
 wherein at least some portion of each of the plurality of stochastic barcodes is single stranded to allow hybridization to a decoding nucleic acid, and 
 wherein decoding comprises two or more sequential hybridizations of decoding nucleic acids to each of the plurality of stochastic barcodes; 
   stochastically barcoding the plurality of targets in the sample by hybridizing the plurality of stochastic barcodes with the plurality of targets to generate stochastically barcoded targets;   identifying the spatial location of each of the plurality of targets by correlating the spatial labels of the plurality of the stochastic barcodes with the spatial locations of the plurality of targets in the sample; and   estimating the number of each of the plurality of targets by determining sequences of the spatial labels and molecular labels of the plurality of the stochastic labels and counting the number of the molecular labels with distinct sequences.   
     
     
       32. The method of claim 31, wherein the sample comprises a plurality of cells, and wherein the plurality of targets is associated with the plurality of cells. 
     
     
       33. The method of claim 31, wherein the sample comprises a tissue, a cell monolayer, fixed cells, a tissue section, or any combination thereof. 
     
     
       34. The method of claim 31, wherein the sample is physically divided during stochastically barcoding the plurality of targets in the sample. 
     
     
       35. The method of claim 31, wherein the spatial locations of the plurality of targets in the sample are on a surface of the sample, inside the sample, subcellularly in the sample, or any combination thereof. 
     
     
       36. The method of claim 31, wherein stochastic barcoding the plurality of targets in the sample is performed on the surface of the sample, subcellularly in the sample, inside the sample, or any combination thereof. 
     
     
       37. The method of claim 31, wherein the plurality of targets comprises ribonucleic acids (RNAs), messenger RNAs (mRNAs), microRNAs, small interfering RNAs (siRNAs), RNA degradation products, RNAs each comprising a poly(A) tail, and any combination thereof. 
     
     
       38. The method of claim 31, further comprising visualizing the plurality of targets in the sample. 
     
     
       39. The method of claim 38, wherein visualizing the plurality of targets in the sample comprises mapping the plurality of targets onto a map of the sample. 
     
     
       40. The method of claim 31, wherein the synthetic particles are beads. 
     
     
       41. The method of claim 40, wherein the beads are silica gel beads, controlled pore glass beads, magnetic beads, dynabeads, sephadex/sepharose beads, cellulose beads, polystyrene beads, hydrogel beads, or any combination thereof. 
     
     
       42. The method of claim 31, wherein the molecular labels of different stochastic barcodes are different from one another. 
     
     
       43. The method of claim 31, wherein the sample is intact during stochastically barcoding the plurality of targets in the sample.