Methods and compositions for sequencing complementary polynucleotides

Abstract

Disclosed herein, inter alia, are substrates, kits, and efficient methods of preparing and sequencing two or more regions of a double-stranded polynucleotide.

1 - 29 . (canceled) 
     
     
         30 . A kit comprising:
 a polynucleotide comprising one or more locked nucleic acid (LNA) nucleotides;   a plurality of nucleotide triphosphates comprising dATP, dCTP, dTTP, dGTP, and dUTP;   a plurality of modified nucleotides;   a polymerase; and   a solid support comprising a plurality of immobilized forward oligonucleotides and a plurality of immobilized reverse oligonucleotides.   
     
     
         31 . The kit of  claim 30 , wherein the polymerase is a strand-displacing polymerase. 
     
     
         32 . The kit of  claim 30 , further comprising an exonuclease. 
     
     
         33 . The kit of  claim 32 , wherein the exonuclease comprises lambda exonuclease. 
     
     
         34 . The kit of  claim 30 , further comprising a cleaving agent. 
     
     
         35 . The kit of  claim 34 , wherein the cleaving agent comprises a DNA glycosylase, endonuclease, or combination thereof. 
     
     
         36 . The kit of  claim 30 , wherein the polynucleotide comprises more than 10%, more than 20%, more than 30%, more than 40%, or more than 50% LNAs. 
     
     
         37 . The kit of  claim 30 , wherein the polynucleotide comprises less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% canonical dNTPs. 
     
     
         38 . The kit of  claim 30 , wherein the polynucleotide comprises SEQ ID NO:1-SEQ ID NO:59. 
     
     
         39 . The kit of  claim 30 , wherein the polynucleotide comprises a plurality of consecutive LNAs. 
     
     
         40 . The kit of  claim 30 , wherein the polynucleotide comprises, from 5′ to 3′, one or more LNAs followed by a plurality of native nucleotides. 
     
     
         41 . The kit of  claim 30 , wherein the polynucleotide is about 15 to about 50 nucleotides in length. 
     
     
         42 . The kit of  claim 30 , wherein the melting temperature (Tm) of the polynucleotide is about 70° C. to about 95° C. 
     
     
         43 . The kit of  claim 30 , wherein the modified nucleotides comprise a detectable label. 
     
     
         44 . The kit of  claim 43 , wherein the modified nucleotides further comprise a 3′ reversible terminator moiety. 
     
     
         45 . The kit of  claim 30 , further comprising a denaturant, wherein said denaturant comprises ethylene glycol, polyethylene glycol, 1,2-propanediol, dimethyl sulfoxide (DMSO), glycerol, formamide, 7-deaza-dGTP, acetamide, betaine, or tetramethylammonium chloride (TMAC). 
     
     
         46 . The kit of  claim 30 , further comprising a plurality of dideoxynucleotide triphosphates (ddNTPs). 
     
     
         47 . The kit of  claim 30 , wherein the polynucleotide further comprises one or more cleavable sites. 
     
     
         48 . The kit of  claim 30 , wherein the plurality of immobilized forward oligonucleotides and reverse oligonucleotides comprise one or more cleavable sites. 
     
     
         49 . A substrate comprising:
 i) a plurality of first polynucleotides attached to the substrate;   ii) a plurality of second polynucleotides attached to the same substrate, wherein the plurality of second polynucleotides comprise a complementary sequence to the plurality of first polynucleotides; and   iii) a plurality of third polynucleotides hybridized to the plurality of second polynucleotides, wherein the plurality of third polynucleotides are not attached to the substrate, and wherein each of the plurality of third polynucleotides comprise one or more locked nucleic acid (LNA) nucleotides.   
     
     
         50 . The substrate of  claim 49 , wherein the plurality of third polynucleotides further comprises one or more cleavable sites. 
     
     
         51 . The substrate of  claim 49 , wherein each of the plurality of third polynucleotides comprises a sequence selected from: SEQ ID NO: 1-SEQ ID NO: 59.