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 for determining an identity of a nucleotide at a position, comprising: a) providing a device configured for measuring color in more than one detector channel, nucleic acid, polymerase, and a mixture of labeled and non-labeled reversible terminators, said labeled reversible terminators comprising a label linked to a nucleotide analogue, said label producing color; b) incorporating, with said polymerase, a first nucleotide analogue from said mixture into said nucleic acid at a position; c) detecting the label of said incorporated first nucleotide analogue in two channels with said device; and d) determining the identity of the incorporated nucleotide at said position.
2. 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.
3. The method of claim 1 , wherein said labeled reversible terminators are labeled with at least one type of fluorescent dye and said detecting of step c) comprising detecting color of said at least one type of fluorescent dye in two channels.
4. The method of claim 3 , wherein said at least one type of fluorescent dye is visible in the yellow channel and the green channel.
5. The method of claim 3 , wherein said at least one type of fluorescent dye is visible in the red channel and the green channel.
6. The method of claim 1 , wherein said nucleic acid comprises primer hybridized to template.