High throughput nucleic acid sequencing by expansion and related methods

Abstract

Nucleic acid sequencing methods and related products and methods for detection and presentation of the same are disclosed. Methods for sequencing a target nucleic acid comprise providing a daughter strand produced by a template-directed synthesis, the daughter strand comprising a plurality of subunits coupled in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of the target nucleic acid.

1 . A method for sequencing a target nucleic acid, comprising:
 a) providing an S-Xdaughter strand produced by a template-directed synthesis, the daughter strand comprising a plurality of subunits coupled in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of the target nucleic acid, wherein the individual subunits comprise a tether, at least one probe, and at least one selectively cleavable bond, the at least one probe comprising X nucleobase residues (with X being a positive integer greater than one) and at least one reporter construct that encodes the genetic information of Y nucleobase residue(s) of the probe (with Y being a positive integer less than X);   b) cleaving the at least one selectively cleavable bond to yield an S-Xpandomer of a length longer than the plurality of the subunits of the S-Xdaughter stand, the S-Xpandomer comprising the tethers and reporter elements for determining Y nucleobase(s) every X nucleobases; and   c) detecting the at least one reporter construct to decode the genetic information of Y nucleobase(s) every X nucleobases of the daughter strand.   
     
     
         2 . The method of  claim 1 , wherein the target nucleic acid is produced by a template-directed rolling circle polymerization process. 
     
     
         3 . The method of  claim 1 , wherein the template directed synthesis comprises a ligation reaction. 
     
     
         4 . The method of  claim 3 , wherein the ligation reaction is an enzymatic ligation reaction. 
     
     
         5 . The method of  claim 1 , wherein the at least one reporter construct is associated with:
 the tethers of the S-Xpandomer;   the S-Xdaughter strand prior to cleavage of the at least one selectively cleavable bond; or   the S-Xpandomer after cleavage of the at least one selectively cleavable bond.   
     
     
         6 . The method of  claim 1 , wherein the at least one reporter construct is attached to the S-Xdaughter strand after template-directed synthesis thereof. 
     
     
         7 . The method of  claim 1 , wherein the tether is attached to the S-Xdaughter strand after template-directed synthesis thereof. 
     
     
         8 . The method of  claim 1 , wherein the S-Xpandomer further comprises all or a portion of the at least one probe. 
     
     
         9 . The method of  claim 8 , wherein the at least one reporter construct is or is associated with the at least one probe. 
     
     
         10 . The method of  claim 1 , wherein the at least one selectively cleavable bond is:
 a covalent bond;   an intra-tether bond;   a bond between or within probes or nucleobase residues of the S-Xdaughter strand; or   a bond between the probes or nucleobase residues of the S-Xdaughter strand and a target template.   
     
     
         11 . The method of  claim 1 , wherein the S-Xpandomer comprises the following structure: 
       
         
           
           
               
               
           
         
         wherein
 T represents the tether; 
 P 1  represents a first probe moiety; 
 P 2  represents a second probe moiety; 
 κ represents the κ th  subunit in a chain of m subunits, where m is an integer greater than three; and 
 α represents a species of a subunit motif selected from a library of subunit motifs, wherein each of the species comprises sequence information of the contiguous nucleotide sequence of a portion of the target nucleic acid; 
 
       
       
         
           
           
               
               
           
         
         wherein
 T represents the tether; 
 P 1  represents a first probe moiety; 
 P 2  represents a second probe moiety; 
 κ represents the κ th  subunit in a chain of m subunits, where m is an integer greater than three; 
 α represents a species of a subunit motif selected from a library of subunit motifs, wherein each of the species comprises sequence information of the contiguous nucleotide sequence of a portion of the target nucleic acid; and 
 χ represents a bond with the tether of an adjacent subunit; 
 
       
       
         
           
           
               
               
           
         
         wherein
 T represents the tether; 
 P 1  represents a first probe moiety; 
 P 2  represents a second probe moiety; 
 κ represents the κ th  subunit in a chain of m subunits, where m is an integer greater than three; 
 α represents a species of a subunit motif selected from a library of subunit motifs, wherein each of the species comprises sequence information of the contiguous nucleotide sequence of a portion of the target nucleic acid; and 
 χ represents a bond with the tether of an adjacent subunit; 
 
       
       
         
           
           
               
               
           
         
         wherein
 T represents the tether; 
 P 1  represents a first probe moiety; 
 P 2  represents a second probe moiety; 
 κ represents the κ th  subunit in a chain of m subunits, where m is an integer greater than three; 
 α represents a species of a subunit motif selected from a library of subunit motifs, wherein each of the species comprises sequence information of the contiguous nucleotide sequence of a portion of the target nucleic acid; and 
 χ represents a bond with the tether of an adjacent subunit; 
 
       
       
         
           
           
               
               
           
         
         wherein
 T represents the tether; 
 κ represents the κ th  subunit in a chain of m subunits, where m is an integer greater than three; 
 α represents a species of a subunit motif selected from a library of subunit motifs, wherein each of the species comprises sequence information of the contiguous nucleotide sequence of a portion of the target nucleic acid; and 
 χ represents a bond with the tether of an adjacent subunit; 
 
       
       
         
           
           
               
               
           
         
         wherein
 T represents the tether; 
 N represents a nucleobase residue; 
 κ represents the κ th  subunit in a chain of m subunits, where m is an integer greater than ten; 
 α represents a species of a subunit motif selected from a library of subunit motifs, wherein each of the species comprises sequence information of the contiguous nucleotide sequence of a portion of the target nucleic acid; and 
 χ represents a bond with the tether of an adjacent subunit; 
 
       
       
         
           
           
               
               
           
         
         wherein
 T represents the tether; 
 κ represents the κ th  subunit in a chain of m subunits, where m is an integer greater than ten; 
 α represents a species of a subunit motif selected from a library of subunit motifs, wherein each of the species comprises sequence information of the contiguous nucleotide sequence of a portion of the target nucleic acid; and 
 χ represents a bond with the tether of an adjacent subunit; 
 
       
       
         
           
           
               
               
           
         
         wherein
 T represents the tether; 
 N represents a nucleobase residue; 
 κ represents the κ th  subunit in a chain of m subunits, where m is an integer greater than ten; 
 α represents a species of a subunit motif selected from a library of subunit motifs, wherein each of the species comprises sequence information of the contiguous nucleotide sequence of a portion of the target nucleic acid; and 
 χ represents a bond with the tether of an adjacent subunit; 
 
       
       
         
           
           
               
               
           
         
         wherein
 T represents the tether; 
 N represents a nucleobase residue; 
 κ represents the κ th  subunit in a chain of m subunits, where m is an integer greater than ten; 
 α represents a species of a subunit motif selected from a library of subunit motifs, wherein each of the species comprises sequence information of the contiguous nucleotide sequence of a portion of the target nucleic acid; 
 χ 1  represents a bond with the tether of an adjacent subunit; and 
 χ 2  represents an inter-tether bond; or 
 
       
       
         
           
           
               
               
           
         
         wherein
 T represents the tether; 
 n 1  and n 2  represents a first portion and a second portion, respectively, of a nucleobase residue; 
 κ represents the κ th  subunit in a chain of m subunits, where m is an integer greater than ten; and 
 α represents a species of a subunit motif selected from a library of subunit motifs, wherein each of the species comprises sequence information of the contiguous nucleotide sequence of a portion of the target nucleic acid. 
 
       
     
     
         12 . The method of  claim 11 , wherein the S-Xdaughter strand is formed from a plurality of oligomer substrate constructs having the following structure: 
       
         
           
           
               
               
           
         
         wherein
 T represents the tether; 
 P 1  represents a first probe moiety; 
 P 2  represents a second probe moiety; 
 ˜ represents the at least one selectively cleavable bond; and 
 R 1  and R 2  represent the same or different end groups for the template directed synthesis of the daughter strand; 
 
       
       
         
           
           
               
               
           
         
         wherein
 T represents the tether; 
 P 1  represents a first probe moiety; 
 P 2  represents a second probe moiety; 
 R 1  and R 2  represent the same or different end groups for the template directed synthesis of the daughter strand; 
 ε represents a first linker group; 
 δ represents a second linker group; and 
 “----” represents a cleavable intra-tether crosslink; 
 
       
       
         
           
           
               
               
           
         
         wherein
 T represents the tether; 
 P 1  represents a first probe moiety; 
 P 2  represents a second probe moiety; 
 R 1  and R 2  represent the same or different end groups for the template directed synthesis of the daughter strand; 
 ε represents a first linker group; 
 δ represents a second linker group; and 
 “----” represents a cleavable intra-tether crosslink; 
 
       
       
         
           
           
               
               
           
         
         wherein
 T represents the tether; 
 P 1  represents a first probe moiety; 
 P 2  represents a second probe moiety; 
 ˜ represents the at least one selectively cleavable bond; 
 R 1  and R 2  represent the same or different end groups for the template directed synthesis of the daughter strand; 
 ε represents a first linker group; and 
 δ represents a second linker group; 
 
       
       
         
           
           
               
               
           
         
         wherein
 T represents the tether; 
 P 1  represents a first probe moiety; 
 P 2  represents a second probe moiety; 
 ˜ represents the at least one selectively cleavable bond; 
 R 1  and R 2  represent the same or different end groups for the template directed synthesis of the daughter strand; 
 ε represents a first linker group; and 
 δ represents a second linker group; 
 
       
       
         
           
           
               
               
           
         
         wherein
 T represents the tether; 
 N represents a nucleobase residue; 
 R 1  and R 2  represent the same or different end groups for the template directed synthesis of the daughter strand; 
 ε represents a first linker group; 
 δ represents a second linker group; and 
 “----” represents a cleavable intra-tether crosslink; 
 
       
       
         
           
           
               
               
           
         
         wherein
 T represents the tether; 
 N represents a nucleobase residue; 
 R 1  and R 2  represent the same or different end groups for the template directed synthesis of the daughter strand; 
 ˜ represents the at least one selectively cleavable bond; 
 ε represents a first linker group; 
 δ represents a second linker group; and 
 “----” represents a cleavable intra-tether crosslink; 
 
       
       
         
           
           
               
               
           
         
         wherein
 T represents the tether; 
 N represents a nucleobase residue; 
 R 1  and R 2  represent the same or different end groups for the template directed synthesis of the daughter strand; 
 ε represents a first linker group; 
 δ represents a second linker group; and 
 “----” represents a cleavable intra-tether crosslink; 
 
       
       
         
           
           
               
               
           
         
         wherein
 T represents the tether; 
 N represents a nucleobase residue; 
 R 1  and R 2  represent the same or different end groups for the template directed synthesis of the daughter strand; 
 ε 1  and ε 2  represent the same or different first linker groups; 
 δ 1  and ε 2  represent the same or different second linker groups; and 
 “----” represents a cleavable intra-tether crosslink; or 
 
       
       
         
           
           
               
               
           
         
         wherein
 T represents the tether; 
 N represents a nucleobase residue; 
 V represents an internal cleavage site of the nucleobase residue; and 
 R 1  and R 2  represent the same or different end groups for the template directed synthesis of the daughter strand. 
 
       
     
     
         13 . The method of  claim 12 , wherein R 1  and R 2  are selected from hydroxyl, phosphate, and triphosphate. 
     
     
         14 . The method of  claim 1 , wherein the target nucleic acid is produced by a rolling circle polymerization process. 
     
     
         15 . A method for sequencing a target nucleic acid, comprising:
 a) providing a paired-end daughter strand produced by a bidirectional template-directed synthesis, the paired-end daughter strand comprising a first and second sequence region joined to a first and second end of a primer region, respectively, each sequence region independently comprising at least 10 nucleobase residues coupled in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of the target nucleic acid;   b) using the paired-end daughter strand as the analyte input to sequence at least 10 nucleobase residues of each of the first and second probe regions to decode the genetic information of the target nucleic acid.   
     
     
         16 - 18 . (canceled) 
     
     
         19 . A method for sequencing a target nucleic acid, comprising:
 a) providing a paired-end surrogate polymer daughter strand produced by a bidirectional template-directed synthesis, the paired-end surrogate polymer daughter strand having a first and second probe region joined to a first and second end of a primer region, respectively, the first and second probe regions comprising a plurality of surrogate polymer substrates coupled in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of the target nucleic acid, wherein the individual surrogate polymer substrates comprise a tether, at least one probe, and at least one selectively cleavable bond, the individual probe comprising X nucleobase residues (with X being a positive integer greater than one) and at least one reporter element that encodes Y nucleobase residue(s) (with Y being a positive integer of at least one and up to a maximum of X);   b) cleaving the at least one selectively cleavable bond to yield a paired-end surrogate polymer of a length longer than the plurality of the surrogate polymer substrates of the daughter stand, the paired-end surrogate polymer comprising the tethers and reporter elements for determining Y nucleobase(s) every X nucleobases; and   c) detecting the reporter elements to determine Y nucleobase(s) every X nucleobases of the paired-end daughter strand.   
     
     
         20 - 22 . (canceled) 
     
     
         23 . A method for producing a paired-end nucleic acid comprising a first and second region joined to a first and second end of a primer region, respectively, wherein the first and second regions independently comprise at least 4 oligonucleotides, the method comprising:
 a) providing a primer adapter, wherein the primer adapter comprises a region complementary, or near complementary, to a primer;   b) providing the primer, wherein the primer comprises a 5′ phosphate end and a 3′ hydroxyl end;   c) duplexing the primer to the primer adapter; and   d) extending the primer from both the 5′ end and the 3′ end, wherein extending comprises ligating at least 4 oligonucleotides to the 5′ end of the primer.   
     
     
         24 - 29 . (canceled) 
     
     
         30 . A paired-end surrogate polymer comprising a first probe region and a second probe region, the first and second probe regions joined to a first and second end of a primer region, respectively, the first and second probe regions comprising a plurality of subunits coupled in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of a target nucleic acid. 
     
     
         31 - 32 . (canceled) 
     
     
         33 . A kit comprising a plurality of constructs, wherein the constructs comprise S-Xmers comprising the appropriate R1/R2 end groups for forming an S-Xdaughter strand by a template-directed synthesis, wherein the kit optionally comprises appropriate instructions for use of the same in forming an S-Xdaughter strand. 
     
     
         34 . (canceled) 
     
     
         35 . A method of reading individual reporter elements of a surrogate polymer, comprising:
 a) providing a surrogate polymer, wherein the surrogate polymer comprises one or more individual reporter elements;   b) providing a detector construct;   b) presenting the surrogate polymer to the detector construct;   c) reading the individual reporter elements sequentially to determine the reporter element sequence; and   d) using the reporter sequence thus determined to decode the genetic information of the surrogate polymer.   
     
     
         36 - 53 . (canceled) 
     
     
         54 . A method of detecting an analyte, comprising:
 a) providing at least one analyte;   b) providing at least one indicator moiety, wherein the indicator moiety is not associated with the analyte;   c) providing a detector construct, wherein the detector construct comprises a first and a second reservoir comprising first and second electrodes, respectively, wherein the first and second reservoirs are separated by a nanopore substrate positioned between the first and second reservoirs;   d) providing an electric potential to the first and second electrodes, wherein the electric potential is sufficient to translocate the at least one analyte and the at least one indicator moiety through the at least one nanopore channel; and   e) detecting a change in an optical signal emitted from the at least one indicator moiety at or near the at least one nanopore channel as the at least one analyte translocates through the at least one nanopore channel.   
     
     
         55 - 73 . (canceled) 
     
     
         74 . A method of presenting at least one surrogate polymer for detection, comprising:
 a) providing a detector construct, wherein the detector construct comprises at least one detector element;   b) providing the at least one surrogate polymer, wherein the at least one surrogate polymer comprises one or more individual reporter elements; and   c) processing the at least one surrogate polymer to obtain a uniform spatial and temporal spacing of the one or more individual reporter elements.   
     
     
         75 - 130 . (canceled)