US2023304078A1PendingUtilityA1

Spatially distinguished, multiplex nucleic acid analysis of biological specimens

87
Assignee: SPATIAL TRANSCRIPTOMICS ABPriority: Apr 10, 2015Filed: May 9, 2023Published: Sep 28, 2023
Est. expiryApr 10, 2035(~8.7 yrs left)· nominal 20-yr term from priority
Inventors:Jonas FrisenPatrik StahlJoakim LundebergGordon M. CannLeila BazarganAlex Aravanis
C12Q 1/6834C12Q 1/6841C12Q 1/6874C12N 15/1065C12Q 1/6876B01L 3/502B01L 3/5055B01L 2300/0877B01L 2300/163
87
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Claims

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.

Claims

exact text as granted — not AI-modified
1 . (canceled) 
     
     
         2 . A composition comprising:
 a plurality of nucleic acid probes on a solid support, wherein the nucleic acid probes are randomly located and attached across a population of features on the solid support, and wherein a nucleic acid probe of the plurality of nucleic acid probes comprises:
 i) a universal primer binding sequence, and 
 ii) a spatial barcode sequence that differs from the spatial barcode sequence of other nucleic acid probes on the solid support. 
   
     
     
         3 . The composition of  claim 2 , wherein the population of features on the solid support is a pattern of discrete features, wherein a feature in the pattern of discrete features is selected from the group consisting of: beads, pits, wells, channels, ridges, raised regions, pegs, and posts, and wherein the features in the pattern of discrete features on the solid support have an average pitch of less than 1 micron. 
     
     
         4 . The composition of  claim 3 , wherein the solid support is a flowcell or a slide. 
     
     
         5 . The composition of  claim 4 , wherein the solid support further comprises fiducial markers. 
     
     
         6 . The composition of  claim 2 , wherein the solid support comprises a gel coating. 
     
     
         7 . The composition of  claim 6 , wherein the nucleic acid probes are attached to the gel coating. 
     
     
         8 . The composition of  claim 2 , wherein the nucleic acid probe further comprises one or more of a capture sequence and a unique molecular identifier sequence. 
     
     
         9 . The composition of  claim 8 , wherein the nucleic acid probe is decoded and further comprises a poly (T) capture sequence that hybridizes to a nucleic acid sequence from a permeabilized biological specimen. 
     
     
         10 . The composition of  claim 9 , wherein the biological specimen is on the surface of the solid support. 
     
     
         11 . The composition of  claim 10 , wherein the nucleic acid sequence from the biological specimen is hybridized to the capture sequence. 
     
     
         12 . The composition of  claim 10 , further comprising a complementary copy of the nucleic acid sequence from the biological specimen that is hybridized to the capture sequence. 
     
     
         13 . The composition of  claim 12 , further comprising a polymerase, wherein the polymerase is a DNA polymerase, an RNA polymerase, or a reverse transcriptase. 
     
     
         14 . The composition of  claim 9 , wherein the biological specimen is a mixture of cells or a tissue section. 
     
     
         15 . The composition of  claim 14 , wherein the tissue section is a fresh-frozen tissue section. 
     
     
         16 . The composition of  claim 14 , wherein the tissue section is a fixed tissue section. 
     
     
         17 . The composition of  claim 16 , wherein the fixed tissue section is a deparaffinized formalin-fixed paraffin-embedded tissue. 
     
     
         18 . The composition of  claim 9 , wherein the biological specimen is from a plant, an algae, an insect, a nematode, a fish, a reptile, a fungi, or a mammal. 
     
     
         19 . The composition of  claim 18 , wherein the mammal is a human. 
     
     
         20 . The composition of  claim 8 , wherein the nucleic acid sequence is RNA. 
     
     
         21 . The composition of  claim 20 , wherein the nucleic acid sequence is mRNA. 
     
     
         22 . The composition of  claim 9 , wherein the nucleic acid sequence is DNA. 
     
     
         23 . The composition of  claim 2 , wherein the universal primer binding sequence is a sequencing primer binding site. 
     
     
         24 . A method for producing a spatial array on a solid support comprising a plurality of nucleic acid probes that are randomly located and attached across a population of features on the solid support, said method comprising:
 a) randomly distributing the plurality of nucleic acid probes on the solid support, wherein a nucleic acid probe of the plurality comprises: (i) a universal primer binding sequence, and (ii) a spatial barcode sequence that differs from the spatial barcode sequence of other nucleic acid probes on the solid support; and   b) decoding the spatial barcode sequence of the plurality of nucleic acid probes attached across the population of features on the solid support, thereby identifying the location of the spatial barcode sequences on the solid support and producing the spatial array.   
     
     
         25 . The method of  claim 24 , wherein the nucleic acid probe further comprises a unique molecular identifier sequence. 
     
     
         26 . The method of  claim 24 , wherein the nucleic acid probe further comprises a capture sequence. 
     
     
         27 . The method of  claim 26 , wherein the capture sequence is a poly(T) capture sequence. 
     
     
         28 . The method of  claim 24 , wherein the spatial array further comprises fiducial markers. 
     
     
         29 . The method of  claim 24 , wherein the population of features on the solid support is a pattern of discrete features, wherein a feature in the pattern of discrete features is selected from the group consisting of: beads, pits, wells, channels, ridges, raised regions, pegs, and posts, and wherein the features in the pattern of discrete features on the solid support have an average pitch of less than 1 micron. 
     
     
         30 . The method of  claim 24 , wherein the solid support is a flow cell or a slide. 
     
     
         31 . The method of  claim 24 , wherein the universal primer binding sequence is a sequencing primer binding site.

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