Phase protective reagent flow ordering

A method for extending a primer hybridized to a nucleic acid template, said method comprising:

• (a) contacting the primer with a first extension solution in the presence of a polymerase;
• (b) contacting the primer with a second extension solution in the presence of a polymerase thereby extending said primer by a single nucleotide;
• wherein:
• (i) said first extension solution comprises a first doublet of nucleotide types and said second extension solution comprises a second doublet of nucleotide types, wherein said first doublet of nucleotide types have no nucleotide types in common with said second doublet of nucleotide types;
• (ii) said first extension solution comprises a first triplet of nucleotide types and said second extension solution comprises a second triplet of nucleotide types, wherein said first triplet of nucleotide types has one or two nucleotide types in common with said second triplet of nucleotide types;
• (iii) said first extension solution comprises a first doublet of nucleotide types and said second extension solution comprises a second triplet of nucleotide types, wherein said first doublet of nucleotide types has one or two nucleotide types in common with said second triplet of nucleotide types;
• or
• (iv) said first extension solution comprises a first triplet of nucleotide types and said second extension solution comprises a second doublet of nucleotide types, wherein said first triplet of nucleotide types has one or two nucleotide types in common with said second doublet of nucleotide types;
• and
• (c) repeating steps (a) and (b), wherein each repetition of steps (a) and (b) is a cycle, wherein each cycle is performed at least 20 times thereby performing a series of cycles, wherein each cycle is a first ordered cycle or a second ordered cycle, wherein said first ordered cycle contacts the primer with said first extension solution first and said second extension solution second, wherein said second ordered cycle contacts the primer with said second extension solution first and said first extension solution second, wherein said series of cycles is performed according to a non-cyclic sequence.

A kit for determining the identity of a base in a target nucleic acid by sequencing-by-synthesis, the kit comprising (a) a first mixture of deoxyribonucleotide triphosphates (dNTPs) comprising:

• a first plurality of dNTPs comprising a first label;
• and a second plurality of dNTPs comprising a second label;
• and (b) a second mixture of deoxyribonucleotide triphosphates (dNTPs) comprising:
• a third plurality of dNTPs comprising the first label;
• and a fourth plurality of dNTPs comprising the second label;
• and (c) a third mixture comprising non-incorporating dNTPs comprising:
• a first plurality of non-incorporating dNTPs;
• a second plurality of non-incorporating dNTPs;
• a third plurality of non-incorporating dNTPs;
• and a fourth plurality of non-incorporating dNTPs;
• wherein each of the first, second, third, and fourth pluralities of dNTPs is selected from the group consisting of dATP, dTTP, dCTP, and dGTP, and are different from each other;
• wherein the first label and the second label are different labels and are distinguishable;
• and wherein each of the first, second, third, and fourth pluralities of non-incorporating dNTPs is selected from the group consisting of a non-incorporable analog of dATP, a non-incorporable analog of dTTP, a non-incorporable analog of dCTP, and a non-incorporable analog of dGTP, and are different from each other.