Methods and compositions for nucleic acid sequencing

A method for determining sequences of a plurality of target polynucleotides on a solid support, comprising performing repeated cycles of:

• (i) DNA polymerase catalyzed incorporation of four different types of nucleotides into the plurality of target polynucleotides, wherein each of the four different types of nucleotide comprises a 3′ blocking group, wherein the four different types of nucleotides are simultaneously present and compete for incorporation into the plurality of target polynucleotides during the DNA polymerase catalyzed incorporation to form extended target polynucleotides, and wherein:
• (a) a first type of nucleotide carries a first label;
• (b) a second type of nucleotide carries a second label;
• (c) a third type of nucleotide is a mixture of a third type of nucleotide carrying the first label or a third label, and a third type of nucleotide carrying the second label or a fourth label;
• wherein the first label and the third label have similar fluorescence emission spectra and form a first dye set, the second label and the fourth label have similar fluorescence emission spectra and form a second dye set, and wherein the two dye sets have different fluorescence emission spectra;
• (d) a fourth type of nucleotide is unlabeled;
• (ii) performing a first imaging event and a second imaging event on the solid support and detecting emission fluorescence in a first channel and a second channel;
• (iii) cleaving the 3′ blocking group from the nucleotides incorporated into the extended target polynucleotides;
• and
• (iv) washing the cleaved 3′ blocking group away from the extended target polynucleotides.