Abstract: Embodiments of the present disclosure relate to kits, compositions, and methods for nucleic acid sequencing, for example, two-channel nucleic acid sequencing by synthesis using blue and green light excitation. In particular, unlabeled nucleotides for incorporation may be used in conjunction with affinity reagents containing detectable labels excitable by blue and/or green lights, for specific binding to each type of nucleotides incorporated.

Abstract: Systems and methods of identifying nucleobases in a template polynucleotide are disclosed. In one embodiment, such a method may include providing a substrate comprising a plurality of the template polynucleotides in a cluster. The method may further include generating light to stimulate fluorescent emissions from the cluster. The method may further include receiving a first signal emitted at a first intensity from a first plurality of nucleotide analogs hybridized to the plurality of template polynucleotides at a first site. The method may further include receiving a second signal emitted at a second intensity from a second plurality of nucleotide analogs hybridized to the plurality of template polynucleotides at a second site. The method may further include identifying the nucleobases hybridized at the first and second sites of the template polynucleotide based on a combination of the first and second signals.