Spatially Distinguished, Multiplex Nucleic Acid Analysis of Biological Specimens

Abstract

A method for spatially tagging nucleic acids of a biological specimen, including steps of (a) providing a solid support comprising different nucleic acid probes that are randomly located on the solid support, wherein the different nucleic acid probes each includes a barcode sequence that differs from the barcode sequence of other randomly located probes on the solid support; (b) performing a nucleic acid detection reaction on the solid support to locate the barcode sequences on the solid support; (c) contacting a biological specimen with the solid support that has the randomly located probes; (d) hybridizing the randomly located probes to target nucleic acids from portions of the biological specimen; and (e) modifying the randomly located probes that are hybridized to the target nucleic acids, thereby producing modified probes that include the barcode sequences and a target specific modification, thereby spatially tagging the nucleic acids of the biological specimen.

1 . A method for spatially tagging target nucleic acids of a biological specimen, comprising:
 (a) attaching a population of nucleic acid probes to a solid support at randomly located positions on the solid support, wherein the nucleic acid probes comprise a (i) target capture sequence and (ii) a spatial tag sequence that differs from spatial tag sequences of other nucleic acid probes in the population;   (b) performing a nucleic acid detection reaction to determine the spatial tag sequences at the randomly located positions on the solid support;   (c) contacting a biological specimen with the nucleic acid probes on the solid support;   (d) hybridizing the target capture sequences of the nucleic acid probes to target nucleic acids from portions of the biological specimen that are proximal to the nucleic acid probes; and   (e) extending the target capture sequences to produce extended probes that comprise sequences from the target nucleic acids, or portions thereof, and the spatial tag sequences, thereby spatially tagging the target nucleic acids of the biological specimen.   
     
     
         2 . The method of  claim 1 , wherein step (a) further comprises amplifying the nucleic acid probes on the solid support, thereby producing nucleic acid clusters having copies of the spatial tag sequence and the target capture sequence at the randomly located positions on the solid support. 
     
     
         3 . The method of  claim 2 , wherein the nucleic acid clusters on the solid support have an average pitch of less than 10 μm and/or an average area of less than 100 μm2. 
     
     
         4 . The method of  claim 1 , wherein the nucleic acid detection reaction is a sequencing reaction or a decoder probe hybridization reaction. 
     
     
         5 . The method of  claim 1 , wherein the solid support comprises an array of beads, wherein the nucleic acid probes are attached to beads in the array, wherein:
 (i) step (a) comprises randomly distributing the beads on the solid support; or   (ii) the solid support comprises wells having dimensions that accommodate no more than a single bead, wherein: the beads are attached to different spatial tag sequences and there are a greater number of spatial tag sequences than number of wells, the solid support comprises at least 1×106 beads, the array of beads has an average pitch of less than 10 μm, and/or the beads have an average diameter of less than 10 μm.   
     
     
         6 . The method of  claim 1 , wherein the solid support comprises a pattern of discrete features. 
     
     
         7 . The method of  claim 1 , wherein the solid support comprises a gel coating, wherein a plurality of nucleic acid primers is attached to the gel coating, wherein a nucleic acid primer of the plurality of nucleic acid primers comprises a universal primer sequence that is common to the nucleic acid primers of the plurality of nucleic acid primers, and wherein the nucleic acid probe comprises a universal primer binding sequence that hybridizes to the universal primer sequence. 
     
     
         8 . The method of  claim 7 , wherein a second plurality of nucleic acid primers is further attached to the gel coating, wherein a nucleic acid primer of the second plurality of nucleic acid primers comprise a second universal primer sequence that is common to the nucleic acid primers of the second plurality of nucleic acid primers, and wherein the nucleic acid probe comprises a second universal primer binding sequence that hybridizes to the second universal primer sequence, and the amplifying comprises bridge amplification. 
     
     
         9 . The method of  claim 1 , wherein different nucleic acid probes of the population of nucleic acid probes comprise different target capture sequences that hybridize to different target nucleic acids from the biological specimen. 
     
     
         10 . The method of  claim 1 , wherein different nucleic acid probes of the population of nucleic acid probes comprise a common target capture sequence, and the common target capture sequence comprises a poly(T) or a poly(A) sequence. 
     
     
         11 . The method of  claim 1 , wherein the method further comprises:
 acquiring an image of the biological specimen in contact with the solid support; and   correlating the determined spatial tag sequences at the randomly located positions on the solid support with locations in the image of the biological specimen.   
     
     
         12 . The method of  claim 11 , wherein the method further comprises:
 removing the extended probes from the solid support; and   determining the sequences of the target nucleic acids or portions thereof, and the spatial tag sequences for the extended probes that have been removed from the solid support.   
     
     
         13 . The method of  claim 12 , wherein determining the sequences of the target nucleic acids, or portions thereof, and the spatial tag sequences for the extended probes that have been removed from the solid support comprises sequencing-by-synthesis. 
     
     
         14 . The method of  claim 1 , wherein the method further comprises:
 removing the extended probes from the solid support; and   attaching the extended probes that have been removed from the solid support to a second solid support.   
     
     
         15 . The method of  claim 1 , wherein the solid support is located in or on a flow cell during step (b), and the solid support is removed from the flow cell during step (c) or the flow cell is opened to expose the solid support during step (c). 
     
     
         16 . The method of  claim 1 , wherein the biological specimen is a mixture of cells, and step (c) further comprises attaching the cells to the solid support and/or lysing the cells to release the target nucleic acids from the cells. 
     
     
         17 . The method of  claim 1 , wherein the biological specimen is a tissue, and step (c) further comprises attaching the tissue to the solid support and/or permeabilizing the tissue to release the target nucleic acids from the tissue. 
     
     
         18 . The method of  claim 1 , wherein the target nucleic acids are selected from the group consisting of mRNA, gDNA, rRNA, and tRNA. 
     
     
         19 . The method of  claim 18 , wherein the target nucleic acids are mRNA. 
     
     
         20 . The method of  claim 2 , wherein the method further comprises, after step (b), digesting the nucleic acid clusters with a restriction enzyme, thereby revealing the target capture sequences.