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 in a nucleotide sequence, comprising: a) providing primers, nucleic acid template molecules having a nucleotide sequence, 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 providing a signal, said signal having an intensity; b) incorporating, with a polymerase, a labeled reversible terminator from said mixture into a primer bound to a nucleic acid template molecule at a position corresponding to a complementary base in said template molecule; c) measuring the intensity of the signal of said label of said incorporated labeled reversible terminator; and d) determining the identity of the 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 measuring of step c) comprising measuring the intensity of said at least one type of fluorescent dye.
4. The method of claim 3 , wherein said intensity is measured in step c) in four channels.
5. The method of claim 4 , wherein said four channels are blue, green, yellow, and red channels.
6. The method of claim 3 , wherein said intensity is measured by a camera.
7. The method of claim 6 , wherein said intensity is solely dependent on the camera exposure and the concentration of said at least one type of fluorescent dye.
8. The method of claim 3 , wherein said intensity is measured in step c) in two channels.
9. The method of claim 8 , wherein said two channels are the green and yellow channels.
10. The method of claim 8 , wherein said two channels are the green and red channels.
11. A method for determining an identity of a nucleotide at a position in a nucleotide sequence, comprising: a) providing a device capable of measuring color in more than one detector channels, primers, nucleic acid template molecules having a nucleotide sequence, 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 capable of being detected in two channels; b) incorporating, with a polymerase, a labeled reversible terminator from said mixture into a primer bound to a nucleic acid template molecule at a position corresponding to a complementary base in said template molecule; c) detecting the label of said incorporated labeled reversible terminator in two channels with said device; and d) determining the identity of the nucleotide at said position.
12. The method of claim 11 , wherein said mixture of labeled and non-labeled reversible terminators is reversibly terminated by an azidomethyl group at the 3′ position.
13. The method of claim 11 , 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 one type of fluorescent dye in two channels.
14. The method of claim 13 , wherein said at least one type of fluorescent dye is visible in the yellow channel and the green channel.
15. The method of claim 13 , wherein said at least one type of fluorescent dye is visible in the red channel and the green channel.
16. A method for determining an identity of a nucleotide at a position in a nucleotide sequence, comprising: a) providing a device capable of measuring intensity in more than one detector channels, primers, nucleic acid template molecules having a nucleotide sequence, 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 providing a signal, said signal having an intensity; b) incorporating, with a polymerase, a labeled reversible terminator from said mixture into a primer bound to a nucleic acid template molecule at a position corresponding to a complementary base in said template molecule; c) detecting the intensity of the label of said incorporated labeled reversible terminator in two channels with said device; and d) determining the identity of the nucleotide at said position.
17. The method of claim 16 , wherein said mixture of labeled and non-labeled reversible terminators is reversibly terminated by an azidomethyl group at the 3′ position.
18. The method of claim 16 , wherein said labeled reversible terminators are labeled with at least one type of fluorescent dye and said detecting of step c) comprising detecting the intensity of one type of fluorescent dye in two channels.
19. The method of claim 16 , further comprising determining the ratio of the intensities in the two channels.