A method for performing a sequencing-by-synthesis reaction, said method comprising the steps of: (a) providing a plurality of immobilized template nucleic acids comprising a plurality of different sequences; (b) annealing oligonucleotide primers to the template nucleic acids wherein the oligonucleotide primers hybridize to predetermined positions on the template nucleic acids; (c) combining the template nucleic acids and primers annealed thereto with a polymerase and four different reversible terminator deoxyribonucleotide triphosphate (dNTPs) each deoxyribonucleotide comprising a nucleobase, a sugar moiety, and a reversible blocking group, wherein N is adenine or an analog thereof, guanine or an analog thereof, thymine or an analog thereof, and cytosine or an analog thereof, wherein at least one of said four different dNTPs is unlabeled, under conditions in which a plurality of the oligonucleotide primers are extended by incorporation of a single reversible terminator deoxyribonucleotide to produce plurality of primer extension products, some of which comprise A or A' incorporated at the 3' terminus, some of which comprise T or T' incorporated at the 3' terminus, some of which comprise G or G' incorporated at the 3' terminus, and some of which comprise C or C' incorporated at the 3' terminus; (d) contacting the plurality of primer extension products with one or more first affinity reagents under conditions wherein each of said one or more first affinity reagents binds to only one of the four different incorporated reversible terminator deoxyribonucleotides, wherein the first affinity reagent binds to the nucleobase and the reversible blocking group of said one of four different incorporated reversible terminator deoxyribonucleotides; (e) detecting the binding of the one or more first affinity reagents, wherein the binding of a first affinity reagent to a primer extension product comprising an incorporated reversible terminator deoxyribonucleotide identifies the nucleobase of the incorporated reversible terminator deoxyribonucleotide and the nucleobase of the complementary deoxyribonucleotide in the template. (a) providing a plurality of immobilized template nucleic acids comprising a plurality of different sequences; (b) annealing oligonucleotide primers to the template nucleic acids wherein the oligonucleotide primers hybridize to predetermined positions on the template nucleic acids; (c) combining the template nucleic acids and primers annealed thereto with a polymerase and four different reversible terminator deoxyribonucleotide triphosphate (dNTPs) each deoxyribonucleotide comprising a nucleobase, a sugar moiety, and a reversible blocking group, wherein N is adenine or an analog thereof, guanine or an analog thereof, thymine or an analog thereof, and cytosine or an analog thereof, wherein at least one of said four different dNTPs is unlabeled, under conditions in which a plurality of the oligonucleotide primers are extended by incorporation of a single reversible terminator deoxyribonucleotide to produce plurality of primer extension products, some of which comprise A or A' incorporated at the 3' terminus, some of which comprise T or T' incorporated at the 3' terminus, some of which comprise G or G' incorporated at the 3' terminus, and some of which comprise C or C' incorporated at the 3' terminus; wherein N is adenine or an analog thereof, guanine or an analog thereof, thymine or an analog thereof, and cytosine or an analog thereof, wherein at least one of said four different dNTPs is unlabeled, under conditions in which a plurality of the oligonucleotide primers are extended by incorporation of a single reversible terminator deoxyribonucleotide to produce plurality of primer extension products, some of which comprise A or A' incorporated at the 3' terminus, some of which comprise T or T' incorporated at the 3' terminus, some of which comprise G or G' incorporated at the 3' terminus, and some of which comprise C or C' incorporated at the 3' terminus; (d) contacting the plurality of primer extension products with one or more first affinity reagents under conditions wherein each of said one or more first affinity reagents binds to only one of the four different incorporated reversible terminator deoxyribonucleotides, wherein the first affinity reagent binds to the nucleobase and the reversible blocking group of said one of four different incorporated reversible terminator deoxyribonucleotides; (e) detecting the binding of the one or more first affinity reagents, wherein the binding of a first affinity reagent to a primer extension product comprising an incorporated reversible terminator deoxyribonucleotide identifies the nucleobase of the incorporated reversible terminator deoxyribonucleotide and the nucleobase of the complementary deoxyribonucleotide in the template.