Methods and compositions for nucleic acid sequencing
The present disclosure provides methods and systems for detecting multiple different nucleotides in a sample. In particular, the disclosure provides for detection of multiple different nucleotides in a sample utilizing fewer detection moieties than the number of nucleotides being detected and/or fewer imaging events than the number of nucleotides being detected.
1 . A method for determining sequences of a plurality of target polynucleotides on a solid support, comprising performing repeated cycles of:
(i) DNA polymerase catalyzed incorporation of four different types of nucleotides into the plurality of target polynucleotides, wherein each of the four different types of nucleotide comprises a 3′ blocking group, wherein the four different types of nucleotides are simultaneously present and compete for incorporation into the plurality of target polynucleotides during the DNA polymerase catalyzed incorporation to form extended target polynucleotides, and wherein:
(a) a first type of nucleotide carries a first label;
(b) a second type of nucleotide carries a second label;
(c) a third type of nucleotide is a mixture of a third type of nucleotide carrying the first label or a third label, and a third type of nucleotide carrying the second label or a fourth label; wherein the first label and the third label have similar fluorescence emission spectra and form a first dye set, the second label and the fourth label have similar fluorescence emission spectra and form a second dye set, and wherein the two dye sets have different fluorescence emission spectra;
(d) a fourth type of nucleotide is unlabeled;
(ii) performing a first imaging event and a second imaging event on the solid support and detecting emission fluorescence in a first channel and a second channel;
(iii) cleaving the 3′ blocking group from the nucleotides incorporated into the extended target polynucleotides; and
(iv) washing the cleaved 3′ blocking group away from the extended target polynucleotides.
2 . The method of claim 1 , wherein the first imaging event and the second imaging event use two light sources having two different wavelengths.
3 . The method of claim 1 , wherein the first channel detects emission fluorescence from the first label and the third label, the second channel detects emission fluorescence from the second label and the fourth label.
4 . The method of claim 3 , wherein incorporation of the first type of nucleotide is determined by a signal state in the first channel, incorporation of the second type of nucleotide is determined by a signal state in the second channel, incorporation of the third type of nucleotide is determined by signal states in both the first channel and the second channel, and incorporation of the fourth type of nucleotide is determined by dark states in both the first channel and the second channel.
5 . The method of claim 1 , wherein one of the first label and the third label has a higher intensity emission fluorescence than the other label, and one of the second label and the fourth label has a higher intensity emission fluorescence than the other label.
6 . The method of claim 1 , wherein the first label and the third label have an emission λ max offset by up to 100 nm and the second label and the fourth label have an emission λ max offset by up to 100 nm.
7 . The method of claim 1 , wherein the first dye set and the second dye set have an emission λ max offset by at least 100 nm.
8 . The method of claim 1 , wherein the third type of nucleotide is a mixture of the third type of nucleotide carrying the first label, and the third type of nucleotide carrying the second label.
9 . The method of claim 1 , wherein the third type of nucleotide is a mixture of the third type of nucleotide carrying the third label, and the third type of nucleotide carrying the fourth label.
10 . The method of claim 1 , wherein the one or more four different types of nucleotides further comprise one or more linker sequences, and the cleavage of the 3′ blocking group also removes any labels from the incorporated nucleotides.
11 . The method of claim 10 , wherein the linker sequence comprises a cleavable linker moiety and a spacer linker moiety.
12 . The method of claim 11 , wherein the cleavable linker moiety comprises one or more cleavable moieties selected from the group consisting of a disulfide, a diol, a diazo, an ester, a sulfone, an azido, an allyl, and a silyl ether.
13 . The method of claim 12 , wherein the cleavable linker moiety comprises an azido moiety.
14 . The method of claim 13 , wherein the linker sequence comprises the structure:
15 . The method of claim 1 , wherein the 3′ blocking group comprises an azido group.
16 . The method of claim 1 , wherein the 3′ blocking group is cleaved by a water soluble phosphine reagent.
17 . The method of claim 1 , wherein the four different types of nucleotides are selected from the group consisting of dATP, dCTP, dGTP and dTTP or dUTP, and non natural nucleotide analogs thereof.
18 . The method of claim 17 , wherein dGTP or the non natural nucleotide analog thereof is unlabeled.
19 . The method of claim 1 , wherein the method comprises at least 50 repeated cycles.
20 . The method of claim 1 , wherein the method comprises at least 100 repeated cycles.