Adaptors for nucleic acid constructs in transmembrane sequencing

Abstract

The invention relates to adaptors for sequencing nucleic acids. The adaptors may be used to generate single stranded constructs of nucleic acid for sequencing purposes. Such constructs may contain both strands from a double stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) template. The invention also relates to the constructs generated using the adaptors, methods of making the adaptors and constructs, as well as methods of sequencing double stranded nucleic acids.

1 . An adaptor for sequencing nucleic acids, which comprises a region of double stranded nucleic acid, wherein at least one end of the region forms one half of a palindromic cleavage site and wherein the adaptor is differentially selectable from another adaptor. 
     
     
         2 - 16 . (canceled) 
     
     
         17 . A kit comprising at least two populations of adaptors as defined in  claim 1 , wherein every adaptor in each population comprises a nucleic acid sequence that is specific for the population. 
     
     
         18 . A nucleic acid construct for use as a sequencing template comprising a double stranded nucleic acid ligated to at least one adaptor according to  claim 1 . 
     
     
         19 - 21 . (canceled) 
     
     
         22 . A method for preparing an adaptor according to  claim 1 , comprising:
 (a) providing two nucleic acids that are (i) capable of hybridizing to one another to form one half of a palindromic cleavage site and (ii) differentially selectable from those of another adaptor; and   (b) contacting the nucleic acids under conditions which allow them to hybridise and thereby preparing an adaptor.   
     
     
         23 - 26 . (canceled) 
     
     
         27 . A method for preparing a sequence construct, comprising:
 (a) providing double stranded nucleic acid;   (b) contacting the double stranded nucleic acid with a pair of Type I and Type II adaptors under conditions which allow the adaptors to ligate to the nucleic acid;   (c) contacting the ligated products with a surface that specifically binds the Type II adaptors and removing any unbound products;   (d) contacting the surface with an enzyme that recognises the complete palindromic cleavage site and removing any unbound products;   (e) cleaving the Type II adaptors;   (f) contacting the soluble products produced in step (e) with a surface that specifically binds the Type I adaptors and removing any unbound products; and   (g) releasing from the surface the products remaining following step (f) and thereby producing a sequencing construct.   
     
     
         28 - 34 . (canceled) 
     
     
         35 . A method of sequencing nucleic acid molecules with improved accuracy, comprising:
 providing said nucleic acid molecules, each of said nucleic acid molecules comprising a double-stranded portion, the double-stranded portion consisting of a forward strand and a reverse strand, wherein the forward strand and the reverse strand are fully complementary to each other;   linking a 3′ end of the forward strand to a 5′ end of the reverse strand of each of said nucleic acid molecules, thereby forming a plurality of template molecules, wherein said 3′ end of the forward strand and said 5′ end of the reverse strand are linked by a linking oligonucleotide;   performing at least one single-molecule sequencing process on each of said plurality of template molecules, wherein the forward strand and the reverse strand in each of said plurality of template molecules are fully complementary to each other, each of said at least one single-molecule sequencing process consisting of sequencing only one single template molecule of said plurality of template molecules, thereby generating a sequence read from each of said plurality of template molecules, said sequence read comprising sequences of both the forward strand and the reverse strand of each of said plurality of template molecules; and   determining the sequence of each of said plurality of template molecules by analyzing said sequence read from each of said plurality of template molecules, including comparing the sequences of the forward strand and the reverse strand from each of said plurality of template molecules, thereby improving sequencing accuracy of said nucleic acid molecules.   
     
     
         36 . The method of  claim 35 , further comprising linking a 5′ end of the forward strand to a 3′ end of the reverse strand, wherein said 5′ end of the forward strand and said 3′ end of the reverse strand are linked by a second linking oligonucleotide, wherein said linking a 5′ end of the forward strand to a 3′ end of the reverse strand occurs prior to said performing at least one single-molecule sequencing process. 
     
     
         37 . The method of  claim 35 , wherein said at least one single-molecule sequencing process comprises contacting each of said plurality of template molecules with a primer sequence that is complementary to a single-stranded portion of said plurality of template molecules. 
     
     
         38 . The method of  claim 35 , wherein said at least one single-molecule sequencing process comprises initiating sequencing at a gap within each of said plurality of template molecules. 
     
     
         39 . The method of  claim 35 , wherein said at least one single-molecule sequencing process includes single-molecule sequencing of the linking oligonucleotide. 
     
     
         40 . The method of  claim 35 , wherein said at least one single-molecule sequencing process is performed using an electrochemical system. 
     
     
         41 . The method of  claim 35 , wherein said at least one single-molecule sequencing process is performed using a nanopore sensor. 
     
     
         42 . The method of  claim 35 , wherein said at least one single-molecule sequencing process is performed using a sequencing by synthesis technology. 
     
     
         43 . The method of claim  8 , wherein said sequencing synthesis technology comprises detecting incorporation of each nucleotide incorporated by a polymerase mediated, template dependent sequencing process during said at least one single-molecule sequencing process. 
     
     
         44 . The method of  claim 35 , wherein said double-stranded portion comprises at least 500 base pairs. 
     
     
         45 . The method of  claim 35 , wherein said, linking oligonucleotide comprises a registration sequence. 
     
     
         46 . The method of  claim 35 , wherein each of said nucleic acid molecules further comprises a single-stranded portion. 
     
     
         47 . The method of  claim 35 , wherein each of said nucleic acid molecules further comprises an overhang region and said linking a 3′ end of the forward strand to a 5′ end of the reverse strand of each of said nucleic acid molecules comprises ligating said linking oligonucleotide to said overhang region on each of said nucleic acid molecules. 
     
     
         48 . A method of generating nucleotide sequence data for nucleic acid sequencing template molecules, comprising:
 providing said nucleic acid sequencing template molecules, each of said nucleic acid sequencing template molecules consisting of: two nucleic acid strands that are fully complementary to each other and are linked at their one end by a connecting nucleic acid that links a 3′ end of one strand of the two nucleic acid strands to a 5′ end of another strand of the two nucleic acid strands;   performing at least one single-molecule sequencing process on each of said nucleic acid sequencing template molecules, each of said at least one single-molecule sequencing process consisting of sequencing only one single template molecule of said nucleic acid sequencing template molecules; and   monitoring said at least one single molecule sequencing process, thereby generating nucleotide sequence data for each of said two nucleic acid strands of each of said nucleic acid sequencing template molecules, and comparing the sequence data of the two nucleic acid strands from the same nucleic acid template molecule of said nucleic acid sequencing template molecules.   
     
     
         49 . The method of  claim 48 , wherein said at least one single-molecule sequencing process is performed using a sequencing by synthesis technology. 
     
     
         50 . The method of  claim 48 , wherein said at least one single-molecule sequencing process is performed using an electrochemical system. 
     
     
         51 . The method of  claim 48 , wherein said at least one single-molecule sequencing process is performed using a nanopore sensor. 
     
     
         52 . The method of  claim 48 , further comprising comparing the nucleotide sequence data of said one strand of the two nucleic acid strands to the nucleotide sequence data of said another strand of the two nucleic acid strands to determine a nucleotide sequence of each of the nucleic acid sequencing templates. 
     
     
         53 . The method of  claim 48 , wherein said connecting nucleic acid is a hairpin or stem-loop oligonucleotide. 
     
     
         54 . A method of sequencing a linear, single nucleic acid template with improved accuracy, comprising:
 providing a nucleic acid molecule comprising a double-stranded nucleic acid segment consisting of a first strand and a second strand, wherein the first strand and the second strand are fully complementary to each other;   ligating a linking oligonucleotide to a 3′ end of said first strand and a 5′ end of said second strand, wherein the linking oligonucleotide connects said first strand to said second strand of said double-stranded nucleic acid segment, thereby forming a linear, single-stranded nucleic acid sequencing template molecule comprising both said first strand and said second strand, wherein the first strand and the second strand of said linear, single-stranded nucleic acid sequencing template molecule are fully complementary to each other;   performing single-molecule sequencing of said linear, single-stranded nucleic acid sequencing template molecule, said single-molecule sequencing consisting of sequencing only one single template molecule of said linear, single-stranded nucleic acid sequencing template molecule, thereby obtaining a sequence read for said linear, single-stranded nucleic acid sequencing template molecule, wherein said sequence read comprises sequences of said first strand and said second strand of said double-stranded nucleic acid segment, and   determining a sequence of the nucleic acid segment by analyzing said sequencing read, including comparing the sequences of said first strand and said second strand from said linear, single-stranded nucleic acid sequencing template molecule, thereby improving sequencing accuracy of said linear, single-stranded nucleic acid sequencing template molecule.   
     
     
         55 . The method of  claim 54 , wherein said performing single-molecule sequencing of said linear, single-stranded nucleic acid sequencing template molecule comprises sequentially detecting nucleotides in said linear, single-stranded nucleic acid sequencing template molecule. 
     
     
         56 . The method of  claim 55 , wherein said sequentially detecting nucleotides is performed using a polymerase mediated template directed sequencing process. 
     
     
         57 . The method of  claim 55 , wherein said sequentially detecting nucleotides is performed using an electrochemical system. 
     
     
         58 . The method of  claim 55 , wherein said sequentially detecting nucleotides is performed using a nanopore sensor. 
     
     
         59 . The method of  claim 54 , wherein said double-stranded nucleic acid segment comprises at least 500 base pairs. 
     
     
         60 . The method of  claim 54 , wherein said linking oligonucleotide comprises a registration sequence. 
     
     
         61 . The method of  claim 54 , wherein said nucleic acid molecule further comprises an overhang region and said ligating a linking oligonucleotide to a 3′ end of said first strand and a 5′ end of said second strand comprises ligating said linking oligonucleotide to said overhang region on said nucleic acid molecule. 
     
     
         62 . A method of sequencing nucleic acids, the method comprising:
 providing said nucleic acids, each nucleic acid comprising a double-stranded template, the double-stranded template having a sense strand and a complementary antisense strand;   covalently linking a 3′ end of the sense strand to a 5′ end of the antisense strand of each nucleic acid, thereby forming nucleic acid constructs that contain both strands of double stranded nucleic acid templates, wherein, for each nucleic acid construct, the 3′ end of the sense strand and the 5′ end of the antisense strand are linked by a linking oligonucleotide;   sequencing the nucleic acid constructs to obtain a sequence read for each nucleic acid construct, wherein the resulting sequence read of each nucleic acid construct comprises sequences of both its sense strand and its antisense strand; and   determining the sequence of each nucleic acid construct by analyzing the resulting sequence read of each nucleic acid construct, including comparing the sequences of the sense strand and the antisense strand from each nucleic acid construct.   
     
     
         63 . The method of  claim 62 , further comprising, prior to sequencing the nucleic acid constructs, linking a 5′ end of the sense strand to a 3′ end of the antisense strand, wherein said 5′ end of the sense strand and said 3′ end of the antisense strand are linked by a second linking oligonucleotide. 
     
     
         64 . The method of  claim 62 , wherein sequencing of each nucleic acid construct comprises performing single molecule sequencing. 
     
     
         65 . A method of sequencing nucleic acids, the method comprising:
 providing said nucleic acids, each nucleic acid comprising a double-stranded template, the double-stranded template having a sense strand and a complementary antisense strand;   covalently linking a 3′ end of the sense strand to a 5′ end of the antisense strand of each nucleic acid, thereby forming nucleic acid constructs that contain both strands of double stranded nucleic acid templates, wherein, for each nucleic acid construct, the 3′ end of the sense strand and the 5′ end of the antisense strand are linked by a linking oligonucleotide;   sequencing the nucleic acid constructs to obtain a sequence read for each nucleic acid construct, wherein the resulting sequence read of each nucleic acid construct comprises sequences of both its sense strand and its antisense strand; and   determining the sequence of each nucleic acid construct by analyzing the resulting sequence read of each nucleic acid construct, including comparing the sequences of the sense strand and the antisense strand from each nucleic acid construct,   wherein sequencing of each nucleic acid construct comprises using a transmembrane pore, wherein optionally:   i) an exonuclease enzyme is used to sequentially detach the nucleotides from the nucleic acid construct, wherein nucleotides are then detected and discriminated by the pore in order of their release, thus reading the sequence of the nucleic acid construct; or   ii) an enzyme is used that pushes or pulls a nucleic acid strand of the nucleic acid construct through the pore in combination with an applied potential, wherein ionic current fluctuates as a nucleotide in the nucleic acid strand passes through the pore, and wherein fluctuations in the current are indicative of the sequence of the strand; or   iii) products of a polymerase in close proximity to the pore are detected as nucleotides are sequentially added to a nucleic acid strand synthesized using the nucleic acid construct as a template.   
     
     
         66 . The method of  claim 62 , wherein the resulting sequence read of each nucleic acid construct further comprises the sequence of the linking oligonucleotide. 
     
     
         67 . The method of  claim 62 , wherein sequencing of each nucleic acid construct is performed using an apparatus configured to produce voltage-driven ionic transport. 
     
     
         68 . The method of  claim 62 , wherein sequencing of each nucleic acid construct is performed using a nanopore. 
     
     
         69 . The method of  claim 64 , wherein sequencing of each nucleic acid construct is performed by detecting products of a polymerase mediated nucleic acid synthesis. 
     
     
         70 . The method of  claim 69 , wherein, as nucleotides are sequentially added by the polymerase, products of the base addition are detected. 
     
     
         71 . The method of  claim 62 , wherein each double-stranded nucleic acid template is at least 500 nucleotides in length. 
     
     
         72 . The method of  claim 62 , wherein the linking oligonucleotide comprises an artificial, identifiable sequence. 
     
     
         73 . The method of  claim 62 , wherein each of said nucleic acids comprises an overhang. 
     
     
         74 . The method of  claim 62 , wherein each said nucleic acid further comprises an overhang and linking a 3′ end of the sense strand to a 5′ end of the antisense strand of each of said nucleic acids comprises ligating said linking oligonucleotide to said overhang region on each of said nucleic acids. 
     
     
         75 . A method of generating nucleotide sequence data for nucleic acids, comprising:
 providing said nucleic acids, each of said nucleic acids consisting of: two nucleic acid strands that are complementary to each other and are linked at their one end by an adaptor nucleic acid that links a 3′ end of one strand of the two nucleic acid strands to a 5′ end of another strand of the two nucleic acid strands;   performing a sequencing process on each of the nucleic acids, each sequencing process consisting of sequencing only one single nucleic acid; and   monitoring the sequencing process, thereby generating nucleotide sequence data for each of the two nucleic acid strands of each of the nucleic acids, and comparing the sequences of the sense strand and the antisense strand of each nucleic acid.   
     
     
         76 . The method of  claim 75 , wherein the sequencing process is performed by detecting products of a polymerase mediated nucleic acid synthesis. 
     
     
         77 . The method of  claim 75 , wherein the sequencing process is performed using an apparatus configured to produce voltage-driven ionic transport. 
     
     
         78 . The method of  claim 75 , wherein the sequencing process is performed is performed using a nanopore. 
     
     
         79 . The method of claim  14 , further comprising comparing the nucleotide sequence of the sense strand to the nucleotide sequence of the antisense to determine a nucleotide sequence of each of the nucleic acid. 
     
     
         80 . The method of claim  14 , wherein the adaptor nucleic acid is a hairpin oligonucleotide. 
     
     
         81 . A method of sequencing a single stranded nucleic acid construct, the method comprising:
 providing a nucleic acid comprising a double-stranded nucleic acid template, the double-stranded nucleic acid template having a sense strand and an antisense strand;   covalently linking a 3′ end of the sense strand to a 5′ end of the antisense strand with a linking oligonucleotide, thereby forming a single-stranded nucleic acid construct comprising the sense strand and the antisense strand;   sequencing the single-stranded nucleic acid construct to obtain a sequence read for the single-stranded nucleic acid construct, wherein the sequence read comprises sequences of the sense strand and the antisense strand; and   determining the sequence of the double-stranded nucleic acid template by analyzing the sequence read for the single-stranded nucleic acid construct, including comparing the sequences of the sense strand and the antisense strand from the single-stranded nucleic acid construct.   
     
     
         82 . The method of  claim 81 , wherein sequencing the single-stranded nucleic acid construct to obtain a sequence read for the single-stranded nucleic acid construct comprises sequentially detecting nucleotides of the single-stranded nucleic acid construct. 
     
     
         83 . The method of  claim 82 , wherein sequencing of each nucleic acid construct is performed by detecting products of a polymerase mediated nucleic acid synthesis, and wherein, as nucleotides are sequentially added by the polymerase, products of the base addition are detected. 
     
     
         84 . The method of  claim 82 , wherein sequencing of each nucleic acid construct is performed using an apparatus configured to produce voltage-driven ionic transport. 
     
     
         85 . The method of  claim 82 , wherein sequencing of each nucleic acid construct is performed using a nanopore. 
     
     
         86 . The method of  claim 81 , wherein each double-stranded nucleic acid template is at least 500 nucleotides in length. 
     
     
         87 . The method of  claim 81 , wherein the linking oligonucleotide comprises an artificial, identifiable sequence. 
     
     
         88 . The method of  claim 81 , wherein each said nucleic acid further comprises an overhang and linking a 3′ end of the sense strand to a 5′ end of the antisense strand of each of said nucleic acids comprises ligating said linking oligonucleotide to said overhang region on each of said nucleic acids.