Method for transposase mediated spatial tagging and analyzing genomic dna in a biological sample

Abstract

The present disclosure relates to materials and methods for spatially analyzing nucleic acids fragmented with a transposase enzyme, optionally complexed to an antibody-binding moiety (e.g., an antibody-binding protein) bound to an antibody for at least one chromatin protein, in a biological sample.

1 . A method for determining location of accessible genomic DNA in a biological sample, the method comprising:
 (a) providing one or more antibodies to the biological sample, wherein an antibody of the one or more antibodies binds to a chromatin protein in the biological sample;   (b) binding a transposome-antibody-binding moiety complex to the one or more antibodies, wherein the transposome-antibody-binding moiety complex comprises:
 (i) a transposase, 
 (ii) an antibody-binding moiety, and 
 (iii) one or more transposon end sequences; 
   (c) generating fragmented genomic DNA comprising the one or more transposon end sequences;   (d) applying a first microfluidic device having multiple first addressing channels to the biological sample, wherein a first addressing channel identifies a first area in the biological sample;   (e) delivering a first probe through the first addressing channel to the first area in the biological sample, wherein the first probe comprises (i) a first ligation region for ligation to a transposon end sequence of the one or more transposon end sequences, (ii) a first address tag that identifies the first area in the biological sample, and (iii) a second ligation region;   (f) applying a second microfluidic device having multiple second addressing channels to the biological sample, wherein a second addressing channel identifies a second area in the biological sample that intersects with the first area; and   (g) delivering a second probe through the second addressing channel to the second area in the biological sample, wherein the second probe comprises: (i) a second address tag that identifies the second area in the biological sample and (ii) a third ligation region, wherein the second probe is coupled to the first probe at the second ligation region and the third ligation region at an intersection between the first area and the second area, thereby generating a ligation product, and wherein the first and second address tags are used to identify the location of the accessible genomic DNA in the biological sample.   
     
     
         2 . The method of  claim 1 , wherein the one or more antibodies comprises the antibody and a secondary antibody that binds to the antibody. 
     
     
         3 . The method of  claim 2 , wherein the transposome-antibody-binding moiety complex binds to the secondary antibody. 
     
     
         4 . The method of  claim 1 , wherein steps (a) and (b) are performed at substantially the same time, and wherein the antibody and the transposome-antibody-binding moiety complex are combined to form a multi-complex. 
     
     
         5 . The method of  claim 1 , wherein step (a), through step (e) are performed at substantially the same time. 
     
     
         6 . The method of  claim 1 , wherein the antibody-binding moiety is protein A, protein G, or functional derivatives thereof. 
     
     
         7 . The method of  claim 1 , wherein the transposase is a Tn5 transposase enzyme, a Mu transposase enzyme, a Tn7 transposase enzyme, a Vibhar species transposase, or functional derivatives thereof. 
     
     
         8 . The method of  claim 1 , wherein the biological sample is permeabilized prior to step (b), wherein permeabilization is chemical permeabilization or enzymatic permeabilization, and wherein the chemical permeabilization utilizes a reagent comprising a detergent, optionally wherein the detergent is one or more of NP-40, polysorbate-20, and digitonin. 
     
     
         9 . The method of  claim 8 , wherein the enzymatic permeabilization utilizes a reagent comprising a protease of the group consisting of a pepsin, a collagenase, a proteinase K, or combinations thereof. 
     
     
         10 . The method of  claim 1 , wherein the transposon end sequence is inserted into the accessible genomic DNA. 
     
     
         11 . The method of  claim 1 , further comprising (i) coupling the first probe to the transposon end sequence or (ii) coupling the second probe to the first probe between the second ligation region and the third ligation region via ligation. 
     
     
         12 . The method of  claim 11 , wherein the ligation comprises enzymatic ligation, wherein the enzymatic ligation utilizes a ligase selected from the group consisting of a PBCV-1 DNA ligase, a  Chlorella  virus DNA ligase, a single-stranded DNA ligase, and a T4 DNA ligase. 
     
     
         13 . The method of  claim 11 , wherein the ligation comprises use of one or more splint oligonucleotides comprising:
 (i) a sequence that hybridizes to a portion of the transposon end sequence; and (ii) a sequence that hybridizes to a portion of the first probe;   or   (iii) a sequence that hybridizes to a portion of the first probe; and (iv) a sequence that hybridizes to a portion of the second probe.   
     
     
         14 . The method of  claim 11 , wherein the ligation comprises chemical ligation. 
     
     
         15 . The method of  claim 1 , wherein the transposon end sequence is at least about 10 nucleotides to about 50 nucleotides long. 
     
     
         16 . The method of  claim 1 , wherein the multiple first addressing channels or multiple second addressing channels is n addressing channels, wherein n is an integer between about 5 and about 100. 
     
     
         17 . The method of  claim 1 , wherein each of the multiple first addressing channels and multiple second addressing channels has a width of about 5 μm to about 500 μm. 
     
     
         18 . The method of  claim 1 , wherein each of the multiple first addressing channels and multiple second addressing channels has a depth of about 5 μm to about 500 μm. 
     
     
         19 . The method of  claim 1 , wherein each of the multiple first addressing channels and multiple second addressing channels has a distance from other multiple addressing channels of about 5 μm to about 2 mm. 
     
     
         20 . The method of  claim 1 , wherein the first probe and/or the second probe further comprises a variable tag region, a sequencing adaptor, or a combination thereof. 
     
     
         21 . The method of  claim 1 , wherein the first probe and/or the second probe comprises a nucleic acid sequence. 
     
     
         22 . The method of  claim 21 , wherein the nucleic acid sequence comprises DNA. 
     
     
         23 . The method of  claim 1 , further comprising amplifying the ligation product. 
     
     
         24 . The method of  claim 23 , wherein the amplifying is performed by polymerase chain reaction. 
     
     
         25 . The method of  claim 1 , wherein the biological sample is a fixed sample, a frozen sample, a fresh sample, or a fresh frozen sample. 
     
     
         26 . The method of  claim 25 , wherein the fixed sample is a formalin fixed paraffin embedded sample. 
     
     
         27 . The method of  claim 1 , wherein (i) the first and second address tags, and (ii) all or part of the ligation product, or a complement thereof are sequenced to determine the location of the accessible genomic DNA. 
     
     
         28 . The method of  claim 1 , further comprising determining a location of one or more additional accessible genomic DNA in the biological sample, wherein the one or more additional accessible genomic DNA is an integer between 100 and 30,000. 
     
     
         29 . The method of  claim 1 , further comprising staining and imaging the biological sample, wherein the staining is selected from immunohistochemistry, immunofluorescence, hematoxylin, eosin, or a combination thereof. 
     
     
         30 . A composition comprising:
 (a) a transposome complex;   (b) one or more antibodies comprising (i) an antibody or (ii) the antibody and a secondary antibody that binds to the antibody; and   (c) an antibody binding moiety,   wherein the antibody binding moiety is simultaneously bound to the one or more antibodies and the transposome complex; and   wherein the antibody is bound, directly or indirectly, to a nuclear protein.