Methods and compositions for incorporating nucleotides
The invention provides methods and compositions, including, without limitation, algorithms, computer readable media, computer programs, apparatus, and systems for determining the identity of nucleic acids in nucleotide sequences using, for example, data obtained from sequencing by synthesis methods. The methods of the invention include correcting one or more phenomena that are encountered during nucleotide sequencing, such as using sequencing by synthesis methods. These phenomena include, without limitation, sequence lead, sequence lag, spectral crosstalk, and noise resulting from variations in illumination and/or filter responses.
1. A method of incorporating labeled nucleotides into nucleic acid, comprising:
a) providing a plurality of nucleic acid template molecules, a polymerase, a cleaving agent, and a mixture of labeled and non-labeled reversible nucleotide analogue terminators, said labeled reversible nucleotide analogue terminators comprising a label linked to a nucleotide analogue, said label producing color, wherein said labeled reversible nucleotide analogues are labeled with at least one type of fluorescent dye; wherein each reversible terminator contains a reversibly removable chemical moiety capping the 3′-OH group;
b) incorporating a first labeled nucleotide analogue with said polymerase;
c) detecting color of said at least one type of fluorescent dye of the incorporated nucleotide analogue in two channels;
d) removing i) said label of the incorporated nucleotide analogue and ii) said reversibly removable chemical moiety capping the 3′-OH group, with said cleaving agent; and
e) incorporating a second nucleotide analogue.
2. The method of claim 1 , wherein said label and said chemical moiety capping the 3′-OH group are removed at the same time.
3. The method of claim 1 , wherein said at least one type of fluorescent dye is visible in the yellow channel and the green channel.
4. The method of claim 1 , wherein said at least one type of fluorescent dye is visible in the red channel and the green channel.
5. The method of claim 1 , wherein said at least one type of fluorescent dye is visible in the blue channel and the red channel.
6. The method of claim 1 , wherein said non-labeled nucleotides are employed in ratios between 10:1 and 100:1 relative to the labeled nucleotides.
7. The method of claim 1 , wherein said mixture of labeled and non-labeled reversible nucleotide analogues is reversibly terminated by a disulfide bond at the 3′ position.
8. The method of claim 1 , wherein said mixture of labeled and non-labeled reversible terminators is reversibly terminated by an azidomethyl group at the 3′ position.