Accelerated sequencing methods
Described herein are methods of generating a coupled sequencing read pair for a polynucleotide, and methods of analyzing the coupled sequencing read pair. The coupled sequencing read pair can be analyzed to detect polynucleotide variants, including at loci that are not directly sequenced within the coupled sequencing read pair. Other analytical methods can include using coupled sequencing read pairs to construct or validate a consensus sequence. The coupled sequencing read pair may be generated for a polynucleotide by generating sequencing data for a first region by extending a primer using labeled nucleotides; further extending the primer through a second region using nucleotides provided in a second region flow order, wherein primer extension through the second region is faster than primer extension through the first region; and generating sequencing data associated with a sequence of a third region of the polynucleotide by further extending the primer using labeled nucleotides.
1. A method of synchronizing sequencing primers within a sequencing cluster, comprising:
hybridizing primers to polynucleotide copies within a sequencing cluster;
extending the primers through a first region of the polynucleotide copies using a mixture of labeled and unlabeled nucleotides according to a first region flow cycle;
extending the primers through a second region of the polynucleotide copies using one or more re-phasing flows without detecting incorporation of nucleotides into the primers during the one or more re-phasing flows, wherein a mixture of at least two different types of nucleotide bases is used in each of the one or more re-phasing flows; and
extending the primers through a third region of the polynucleotide copies using labeled nucleotides according to a third region flow cycle, wherein only a single type of nucleotide base is used at a time during a flow of the third region flow cycle.
2. The method of claim 1 , wherein a mixture of three different types of nucleotide bases are used in at least one of the one or more re-phasing flows.
3. The method of claim 1 , wherein a mixture of three different types of nucleotide bases are used in each of the one or more re-phasing flows.
4. The method of claim 1 , wherein the one or more re-phasing flows comprises four or more flow steps.
5. The method of claim 4 , wherein a mixture of three different types of nucleotide bases are used in at least one of the one or more re-phasing flows.
6. The method of claim 5 , wherein a mixture of three different types of nucleotide bases are used in each of the one or more re-phasing flows.
7. The method of claim 4 , wherein the one or more re-phasing flows comprises, in any order:
(i) a first flow comprising a mixture comprising A, C, and G nucleotides and omitting T nucleotides;
(ii) a second flow comprising a mixture comprising T, C, and G nucleotides and omitting A nucleotides;
(iii) a third flow comprising a mixture comprising T, A, and G nucleotides and omitting C nucleotides; and
(iv) a fourth flow comprising a mixture comprising T, A, and C nucleotides and omitting G nucleotides.
8. The method of claim 1 , wherein the one or more re-phasing flows comprises one or more of, in any order, (i) a flow comprising a mixture comprising A and C nucleotides and omitting G and T nucleotides; (ii) a flow comprising a mixture comprising T and G nucleotides, and omitting A and C nucleotides; (iii) a flow comprising a mixture comprising A and G nucleotides and omitting T and C nucleotides; (iv) a flow comprising a mixture comprising T and C nucleotides and omitting A and G nucleotides; (v) a flow comprising a mixture comprising A and T nucleotides and omitting G and C nucleotides; (vi) a flow comprising a mixture comprising C and G nucleotides and omitting A and T nucleotides; (vii) a flow comprising a mixture comprising A, G, and C nucleotides and omitting T nucleotides; (viii) a flow comprising a mixture comprising T, A, and G nucleotides and omitting C nucleotides; (ix) a flow comprising a mixture comprising C, T, and A nucleotides and omitting G nucleotides; or (x) a flow step comprising a mixture of G, C, and T nucleotides and omitting A nucleotides.
9. The method of claim 1 , wherein at least one of the one or more re-phasing flows comprises a mixture of A, C, G, and T nucleotides, wherein three base types are non-terminating nucleotides and one base type comprises a reversible terminator.
10. The method of claim 9 , wherein the one or more re-phasing flows comprises (i) a flow comprising a mixture comprising non-terminating A nucleotides, non-terminating C nucleotides, non-terminating G nucleotides, and T nucleotides comprising a reversible terminator; (ii) a flow comprising a mixture comprising non-terminating T nucleotides, non-terminating A nucleotides, non-terminating C nucleotides, and G nucleotides comprising a reversible terminator; (iii) a flow comprising a mixture comprising non-terminating G nucleotides, non-terminating T nucleotides, non-terminating A nucleotides, and C nucleotides comprising a reversible terminator; or (iv) a flow comprising a mixture comprising non-terminating C nucleotides, non-terminating G nucleotides, non-terminating T nucleotides, and A nucleotides comprising a reversible terminator.
11. The method of claim 1 , comprising generating sequencing data associated with a sequence of the first region by detecting the presence or absence of an incorporated labeled nucleotide while extending the primers through the first region.
12. The method of claim 11 , comprising generating sequencing data associated with a sequence of the third region by detecting the presence or absence of an incorporated labeled nucleotide while extending the primers through the third region.
13. The method of claim 1 , comprising generating sequencing data associated with a sequence of the third region by detecting the presence or absence of an incorporated labeled nucleotide while extending the primers through the third region.
14. The method of claim 1 , wherein at least a portion of the nucleotides used to extend the primers through the second region are unlabeled nucleotides.
15. The method of claim 1 , wherein the nucleotides used to extend the primers through the second region are unlabeled nucleotides.
16. The method of claim 1 , wherein the sequencing cluster is attached to a bead or a flowcell.
17. The method of claim 1 , wherein the polynucleotide copies within the sequencing cluster are substantially identical to each other.
18. The method of claim 1 , wherein the sequencing cluster is formed by bridge amplification or emulsion PCR.
19. The method of claim 1 , wherein the sequencing cluster is formed on a bead by emulsion PCR, and wherein the bead is further attached to a surface.