Single-stranded splint strands and methods of use
The present disclosure provides compositions comprising nucleic acid single-stranded splint strands, including kits, and methods that employ the single-stranded splint strands. The single-stranded splint strands can hybridize to portions of linear library molecules to form circularized library-splint complexes having a nick, where the nick can be ligated to form covalently closed circular molecules which can be subjected to downstream amplification and sequencing workflows.
1. A method comprising:
a) providing a plurality of linear single-stranded nucleic acid library molecules ( 100 ) wherein individual linear single-stranded nucleic acid library molecules in the plurality comprise regions arranged in a 5′ to 3′ order: (i) a first left universal adaptor sequence ( 120 ) having a binding sequence for a first surface primer immobilized to a support; (ii) a second left universal adaptor sequence ( 140 ) having a binding sequence for a first sequencing primer; (iii) a sequence of interest ( 110 ); (iv) a second right universal adaptor sequence ( 150 ) having a binding sequence for a second sequencing primer; and (v) a first right universal adaptor sequence ( 130 ) having a binding sequence for a second surface primer immobilized to a support;
b) providing a plurality of single-stranded splint strands ( 200 ) wherein individual single-stranded splint strands ( 200 ) in the plurality comprise regions arranged in a 5′ to 3′ order (i) a first region ( 210 ) having a universal binding sequence that hybridizes with the first left universal adaptor sequence ( 120 ) of individual linear single-stranded nucleic acid library molecules ( 100 ), and (ii) a second region ( 220 ) having a universal binding sequence that hybridizes with the first right universal adaptor sequence ( 130 ) of individual linear single-stranded nucleic acid library molecules ( 100 );
c) hybridizing the plurality of single-stranded splint strands ( 200 ) with the plurality of linear single-stranded nucleic acid library molecules ( 100 ) wherein the first region ( 210 ) of the single-stranded splint strand ( 200 ) hybridizes to the first left universal adaptor sequence ( 120 ) of the linear single-stranded nucleic acid library molecule ( 100 ), and the second region ( 220 ) of the single-stranded splint strand ( 200 ) hybridizes to the first right universal adaptor sequence ( 130 ) of individual linear single-stranded nucleic acid library molecules ( 100 ), thereby forming a plurality of library-splint complexes ( 300 ) comprising circular library molecules having a nick between the terminal 5′ and 3′ ends of individual circular library molecules, wherein the nick is enzymatically ligated to generate a plurality of covalently closed circular library molecules ( 400 );
d) distributing the plurality of covalently closed circular library molecules ( 400 ) onto a support having a plurality of said second surface primers immobilized on the support, and hybridizing individual covalently closed circular library molecules ( 400 ) to individual immobilized second surface primers, thereby immobilizing the plurality of covalently closed circular library molecules ( 400 ) to the support,
wherein the plurality of second surface primers on the support are located at pre-determined or random locations on the support;
e) conducting a rolling circle amplification reaction on the support using the plurality of second surface primers as immobilized amplification primers and the plurality of covalently closed circular library molecules ( 400 ) as template molecules, thereby generating a plurality of single-stranded immobilized nucleic acid concatemer molecules comprising two or more tandem copies of the sequence of interest; and
f) sequencing the sequences of interest of individual single-stranded immobilized nucleic acid concatemer molecules.
2. The method of claim 1 , wherein the plurality of linear single-stranded nucleic acid library molecules ( 100 ) further comprises a first left index sequence ( 160 ) and/or a first right index sequence ( 170 ).
3. The method of claim 1 , wherein the plurality of linear single-stranded nucleic acid library molecules ( 100 ) further comprise a first left unique identification sequence ( 180 ) and/or a first right unique identification sequence ( 190 ).
4. The method of claim 1 , wherein the plurality of covalently closed circular library molecules ( 400 ) of c) are each hybridized to the single-stranded splint strands ( 200 ).
5. The method of claim 4 , further comprising: contacting the plurality of covalently closed circular library molecules ( 400 ) of c) with at least one exonuclease enzyme to remove the plurality of single-stranded splint strands ( 200 ) and retaining the plurality of covalently closed circular library molecules ( 400 ).
6. The method of claim 1 , wherein conducting the rolling circle amplification reaction at step e) comprises contacting the plurality of immobilized covalently closed circular library molecules ( 400 ) with a plurality of strand-displacing polymerases and a plurality of nucleotides.
7. The method of claim 6 , wherein the plurality of nucleotides comprises dATP, dGTP, dCTP, dTTP and/or dUTP.
8. The method of claim 6 , wherein the density of the plurality of single-stranded immobilized nucleic acid concatemer molecules on the support is 10 4 -10 8 per mm 2 .
9. The method of claim 1 , wherein the plurality of second surface primers on the support is in fluid communication with each other to permit flowing a solution of reagents onto the support so that the plurality of immobilized second surface primers can be simultaneously reacted with the reagents in a massively parallel manner, and wherein the solution of reagents comprises nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers or a combination thereof.
10. The method of claim 1 , wherein the sequencing at step f) comprises:
a) contacting the plurality of single-stranded immobilized nucleic acid concatemer molecules with (i) a plurality of sequencing polymerases and (ii) a plurality of soluble sequencing primers, thereby forming a plurality of complexed polymerases each comprising a sequencing polymerase bound to a nucleic acid duplex wherein the nucleic acid duplex comprises a single-stranded immobilized nucleic acid concatemer molecule hybridized to a soluble sequencing primer;
b) contacting the plurality of complexed sequencing polymerases with a plurality of nucleotides thereby binding at least one nucleotide to a complexed sequencing polymerase, wherein the plurality of nucleotides comprises at least one nucleotide analog labeled with a fluorophore and having a removable chain terminating moiety at the sugar 3′ position;
c) incorporating at least one nucleotide into the 3′ end of the hybridized sequencing primers thereby generating a plurality of nascent extended sequencing primers; and
d) detecting the incorporated nucleotide and identifying the nucleo-base of the incorporated nucleotide.
11. The method of claim 10 , wherein the plurality of nucleotides comprises a removable chain terminating moiety at the 3′ sugar group, wherein the removable chain terminating moiety comprises an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, azido group, O-azidomethyl group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group, and wherein the removable chain terminating moiety is cleavable with a chemical compound to generate an extendible 3′OH moiety on the sugar group.
12. The method of claim 10 , wherein the plurality of nucleotides comprises one type of nucleotide selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP.
13. The method of claim 10 , wherein the plurality of nucleotides comprises a mixture of any combination of two or more types of nucleotides selected from a group consisting of dATP, dGTP, dCTP, dTTP and/or dUTP.
14. The method of claim 1 , wherein the support comprises a glass or plastic substrate.
15. The method of claim 1 , wherein the support is passivated with at least one hydrophilic polymer coating having a water contact angle of no more than 45 degrees.
16. The method of claim 15 , wherein the at least one hydrophilic polymer coating comprises a molecule selected from a group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxylethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, and dextran.