Method and apparatus for encoding cellular spatial position information

Abstract

A system, methods, and apparatus are described to collect and prepare single cells and groups of cells from microsamples of specimens and encode spatial information of the physical position of the cells in the specimen. In some embodiment, beads or surfaces with oligonucleotides containing spatial barcodes are used to analyze DNA or RNA. The spatial barcodes allow the position of the cell to be defined and the nucleic acid sequencing information, such as target sequencing, whole genome, gene expression, used to analyze the cells in a microsample for cell type, expression pattern, DNA sequence, and other information, in the context of the cell's physical position in the specimen. In other embodiment, markers such as isotopes are added to a microsample to encode spatial position with mass spectoscopy or other analysis. The spatial encoded information is then readout by analysis such as DNA sequencing, mass spectrometry, fluorescence, or other methods.

1 .- 39 . (canceled) 
     
     
         40 . A method comprising:
 providing a biological specimen;   collecting microsamples from each of a plurality of different spatial positions in the biological specimen;   attaching to nucleic acids in each microsample a marker comprising a nucleic acid barcode comprising spatial information that encodes the original spatial position of the microsample within the biological specimen, thereby producing spatial encoded nucleic acids;   sequencing the spatial encoded nucleic acids; and   based on the spatial information attached to each spatial encoded nucleic acids, determining the original spatial location of the nucleic acid in the biological specimen.   
     
     
         41 .- 42 . (canceled) 
     
     
         43 . A spatial preparation system configured to entrain in a fluidic stream a plurality of microsamples from a biological specimen, wherein the microsamples are contained in spatially separated microdrops in a fluidic stream and positioned in an order based on their original spatial position within the biological specimen, wherein the system comprises:
 a) a spatial sampler subsystem configured to extract a plurality of microsamples from different original spatial positions in a biological specimen; and   b) a spatial encoder subsystem comprising one or more spatial encoder microchannels, each having an inlet and an outlet;   wherein the spatial sampler subsystem delivers the microsamples to the spatial encoder microchannel inlets in a predetermined order based on their original spatial position in the biological specimen, and the spatial encoder subsystem incorporates the microsamples into spatially separated microdrops in a fluidic stream.   
     
     
         44 . The spatial preparation system of  claim 43 , wherein:
 (i) the spatial sampler subsystem comprises:
 (1) a specimen holder, and 
 (2) a multifunctional head comprising a transfer head comprising one or more extraction channels, wherein the extraction channels communicate with a liquid source and, optionally, a gas source, each under positive and/or negative pressure, and wherein the one or more extraction channels comprise ends covered with one or more air permeable, cell impermeable transfer membranes, and wherein, the multifunctional head is mounted on a three axis stage to position the multifunctional head to extract, by contact adhesion or by vacuum, the microsamples from the specimen holder onto the one or more transfer membranes; and 
   (ii) the spatial encoder subsystem comprises:
 (1) a microdroplet generator comprising a source of immiscible liquid in communication with each spatial encoder microchannel at a junction, wherein mixture of the immiscible liquid with the fluidic stream at the junction forms spatially separated microdrops comprising the microsamples; and 
 (2) optionally, a microsample encoder assembly comprising a plurality of reservoirs, each comprising a different spatial marker and each communicating with the spatial encoder microchannel, and, optionally reservoirs comprising a reactants sufficient to attach the tags to analytes in the microsamples, wherein different spatial markers are incorporated with microsamples in different microdrops. 
   
     
     
         45 . The system of  claim 44 , wherein the multifunctional head further comprises a dispense head configured to dispense liquids, e.g., imaging reagents or dissociation solution, onto the biological specimen. 
     
     
         46 . The system of  claim 44 , wherein the transfer head comprises a plurality of extraction channels where in the extraction channels are arrayed in a two-dimensional array (e.g., a line) or a three-dimensional array (e.g., a plane). 
     
     
         47 . The system of  claim 46 , wherein the spatial encoder subsystem comprises a plurality of fluidic channels that merge into the encoder channel in which each has an inlet configured to receive the microsamples from an extraction channel. 
     
     
         48 . The system of  claim 47 , wherein, the transfer membranes have attached thereto a plurality of capture elements, each capture element comprising a particle, which is optionally paramagnetic, having attached thereto one or more antibodies that bind into cells in the biological specimen, and nucleic acid markers comprising positional barcodes comprising spatial information where the spatial information calling to the position of the particle on the multifunctional head. 
     
     
         49 . The system of  claim 48 , wherein, the nucleic acid markers further comprise cell markers identifying the cell to which particle binds, and/or molecular barcodes that differently label different nucleic acid molecules and a single cell. 
     
     
         50 . The system of  claim 44  comprising:
 a) a spatial preparation subsystem of  claim 44 , and 
 b) a spatial librarian subsystem configured to perform a series of biochemical reactions on an emulsion comprising microdrops produced by the spatial preparation subsystem, wherein the spatial librarian subsystem comprises: 
 c) a reaction device comprising an inlet configured to receive microdrops from the spatial preparation subsystem, at least one reaction chamber, and an outlet; 
 d) a reagent rail communicating with the reaction device through a microchannel and comprising reagent sufficient to perform at least one of biochemical reaction on analytes in the microdrops; and 
 e) one or more pumps configured to move the reagents from the reagent rail through the microchannel to the reaction chamber of the reaction device. 
 
     
     
         51 . The spatial analysis system of  claim 50 , wherein the spatial librarian subsystem further comprising:
 f) a temperature controller configured to control temperature in the reaction chamber.   
     
     
         52 . The spatial analysis system of  claim 50 , wherein the spatial librarian subsystem further comprising:
 f) a magnet configured to reversibly immobilize paramagnetic particles contained in the reaction chamber.   
     
     
         53 . The spatial analysis system of  claim 50 , wherein the biochemical reactions comprise at least:
 (i) reverse transcription of messenger RNA into cDNA; and   (ii) amplification of cDNA.   
     
     
         54 . The spatial analysis system of  claim 50 , wherein the biochemical reactions comprise at least:
 (i) primer extension of a primer hybridized to a DNA template to create an extension product; and   (ii) amplification of the extension product.   
     
     
         55 . A method comprising entraining in a fluidic stream a plurality of microsamples from a biological specimen, wherein the microsamples are contained in spatially separated microdrops in the fluidic stream and positioned in an order based on their original spatial position within the biological specimen. 
     
     
         56 . The method of  claim 55 , comprising:
 a) providing a biological specimen;   b) collecting microsamples from each of a plurality of different spatial positions in the biological specimen;   c) introducing the microsamples in a predetermined order into a fluidic stream in a fluidic channel;   d) dividing the fluidic stream into microdrops by introducing boluses of immiscible liquid into the fluidic channel, whereby the microsamples are incorporated into microdrops that are spatially separated from each other in the fluidic stream.   
     
     
         57 . The method of  claim 55 , further comprising:
 (i) introducing into the fluidic stream a plurality of different spatial markers encoding spatial information, wherein the different spatial markers are incorporated into different microdrops in the fluidic stream, thereby encoding each microdrop with spatial information.   
     
     
         58 . The method of  claim 57 , wherein the analytes comprise nucleic acids and the spatial markers comprise nucleic acids comprising nucleic acid barcodes, wherein the method further comprises:
 (e) combining microdrops in a container in the form of an emulsion;   (f) generating spatially tagged nucleic acids by tagging nucleic acid analytes with the nucleic acid barcodes;   (g) breaking the emulsion;   (h) amplifying the tagged nucleic acids.   
     
     
         59 . The method of  claim 58 , wherein the analytes comprise polyadenylated mRNA and the nucleic acid markers further comprise polyT tail, and generating spatially tagged nucleic acids comprises:
 hybridizing the polyT tail to polyadenylated mRNA nucleic acid markers to the mRNA molecules barcodes and reverse transcribing the polyadenylated messenger RNA to produce that spatially tagged cDNA molecules;   performing second strand synthesis on the spatially tagged cDNA molecules to produce tagged double stranded cDNA molecules.   
     
     
         60 . The method of  claim 58 , wherein the analytes comprise DNA molecules and the nucleic acid markers further comprise a nucleotide sequence complementary to a target sequence, and generating spatially tagged nucleic acids comprises:
 hybridizing the complementary nucleotide sequence to a target sequence in the nucleic acid molecules and extending the nucleic acid markers to produce a double-stranded DNA molecule.   
     
     
         61 . The method of  claim 57 , wherein further comprising applying imaging reagent to the biological sample; imaging the biological sample to which the imaging reagent has been applied; based on the imaging selecting features of interest at predetermined spatial positions in the biological sample; and extracting the microsamples including the selected features of interest. 
     
     
         62 . (canceled)