Methods and systems for nucleic acid sequencing

A method for determining a sequence of a template nucleic acid molecule, comprising:

• (a) generating a reaction mixture comprising said template nucleic acid molecule, a primer capable of hybridizing to said template nucleic acid molecule, and a plurality of free nucleotide analogs, wherein a given free nucleotide analog of said plurality of free nucleotide analogs comprises a functional group;
• (b) subjecting said reaction mixture to conditions sufficient to conduct a primer extension reaction on said template nucleic acid molecule in presence of said primer, to incorporate said given free nucleotide analog comprising said functional group into a growing nucleic acid strand having sequence complementarity with said template nucleic acid molecule;
• (c) upon incorporating said given free nucleotide analog into said growing nucleic acid strand, reacting said functional group with a labeling reagent comprising a label to generate a labeled functional group;
• (d) detecting one or more signals indicative of said labeled functional group;
• and
• (e) subjecting said labeled functional group to conditions sufficient to convert said labeled functional group to a moiety that is substantially unreactive with said labeling reagent.