Methods and compositions for incorporating nucleotides

A spectral crosstalk identification method for identifying spectral crosstalk associated with at least one detectable fluorescent label upon incorporation of a nucleotide analogue in a nucleic acid molecule, comprising:

• providing:
• a fluorescence measuring device configured for measuring fluorescence in a plurality of detector channels,
• at least one flow cell including a plurality of spots, each spot comprising a plurality of nucleic acid molecules,
• a solution comprising a mixture including a plurality of nucleotide analogues comprising a plurality of first nucleotide analogues including at least one detectable fluorescent label, and a plurality of second nucleotide analogues without a detectable label, wherein each of the nucleotide analogues in the mixture includes a reversible terminator chemical moiety at the 3′-OH group;
• and
• a polymerase,
• incorporating at least one first nucleotide analogue from the mixture into at least one nucleic acid molecule of at least one spot of the at least one flow cell,
• washing remaining, non-incorporated nucleotide analogues from the at least one flow cell,
• emitting light to cause fluorescence of the at least one detectable fluorescent label of the first nucleotide analogue incorporated in the at least one nucleic acid molecule,
• detecting a first fluorescence intensity of the at least one detectable fluorescent label from the at least one nucleic acid molecule in a first detector channel,
• detecting a second fluorescence intensity of the at least one detectable fluorescent label from the at least one nucleic acid in a second detector channel,
• and
• determining at least one spectral crosstalk factor associated with the at least one detectable fluorescent label in at least one of the first detector channel and the second detector channel, wherein the spectral crosstalk factor is determined based on the contribution of at least one of the first fluorescence intensity and the second fluorescence intensity.