Methods and systems for phasing sequencing strands and long-range sequencing
Described herein are methods synchronizing sequencing primers within a sequencing cluster and methods of generating long-range sequencing reads. The methods can include hybridizing primers to polynucleotide copies within a sequencing cluster; extending the primers through a first region of the polynucleotide copies using labeled nucleotides according to a sequencing flow order; extending the primers through a second region of the polynucleotide copies using one or more re-phasing flow steps that each include at least two different types of nucleotide bases; and extending the primers through a third region of the polynucleotide copies using labeled nucleotides according to the sequencing cycle. The rephasing flow steps may be initiated after a predetermined number of sequencing flow steps, after a measured sequencing signal strength falls below a predetermined sequencing signal strength threshold, or a measured sequencing signal-to-noise ratio falls below a sequencing signal-to-noise ratio threshold.
1 . A method of synchronizing sequencing in a colony, comprising:
(a) providing a sequencing colony comprising a plurality of copies of nucleic acid molecules each having sequence identity to a polynucleotide derived from a sample, wherein each nucleic acid molecule of the plurality of copies comprises a first region, a second region, and a third region;
(b) hybridizing a plurality of primers to the plurality of copies;
(c) extending the plurality of primers through the first region by, in each flow step of a plurality of first flow cycles having a first predetermined number of flow steps, providing a first plurality of nucleotides of a single base type, wherein at least a portion of the first plurality of nucleotides is labeled;
(d) extending, after the first predetermined number of flow steps, the plurality of primers through the second region by, in each flow step of a plurality of second flow cycles, providing a second plurality of nucleotides comprising at least two base types; and
(e) extending the plurality of primers through the third region by, in each flow step of a plurality of third flow cycles, providing a third plurality of nucleotides of a single base type, wherein at least a portion of the third plurality of nucleotides is labeled.
2 . The method of claim 1 , wherein the first predetermined number of flow steps is between about 40 and about 500.
3 . The method of claim 1 , wherein the first predetermined number of flow steps is associated with a predetermined sequencing signal threshold.
4 . The method of claim 1 , wherein the first predetermined number of sequencing flow steps is associated with an expected length of the first region.
5 . The method of claim 1 , wherein the plurality of third flow cycles has a second predetermined number of flow steps, the method further comprising:
(f) extending, after the second predetermined number of flow steps, the plurality of primers through a fourth region of the plurality of copies by, in each flow step of a plurality of fourth flow cycles, providing a fourth plurality of nucleotides comprising at least two base types; and
(g) extending the plurality of primers through a fifth region of the plurality of copies by, in each flow step of a plurality of fifth flow cycles, providing a fifth plurality of nucleotides of a single base type, wherein at least a portion of the fifth plurality of nucleotides is labeled.
6 . The method of claim 5 , wherein the first predetermined number of flow steps and the second predetermined number of flow steps are the same.
7 . The method of claim 1 , wherein a mixture of three different base types is used in at least one flow step of the plurality of second flow cycles.
8 . The method of claim 1 , wherein one or more flow steps of the plurality of second flow cycles comprise 2 to 12 re-phasing flow steps.
9 . The method of claim 1 , wherein one or more flow steps of the plurality of second flow cycles comprise one or more of the following in any order:
(i) a flow step comprising a mixture comprising A, C, and G nucleotides and omitting T nucleotides;
(ii) a flow step comprising a mixture comprising T, C, and G nucleotides and omitting A nucleotides;
(ii) a flow step comprising a mixture comprising T, A, and G nucleotides and omitting C nucleotides; and
(iv) a flow step comprising a mixture comprising T, A, and C nucleotides and omitting G nucleotides.
10 . The method of claim 1 , further comprising sequencing the first region by detecting the presence or absence of an incorporated labeled nucleotide while extending the primers through the first region.
11 . The method of claim 1 , wherein at least a portion of nucleotides provided in the plurality of second flow cycles are unlabeled.
12 . The method of claim 1 , wherein a first flow order of the plurality of first flow cycles and a third flow order of the plurality of third flow cycles are the same.
13 . The method of claim 1 , wherein a first flow order of the plurality of first flow cycles and a third flow order of the plurality of third flow cycles are different.
14 . The method of claim 1 , wherein the plurality of primers is extended through the first region by repeating a first flow order a plurality of times in the plurality of first flow cycles.
15 . The method of claim 14 , wherein the first flow order is repeated 2 times to about 50 times.
16 . The method of claim 1 , wherein a distance between a start of the first region and an end of a final region of the plurality of copies for which sequencing data is generated is at least 300 bases in length.
17 . The method of claim 1 , wherein the sequencing colony is produced by rolling circle amplification of a template nucleic acid molecule, and the sequencing colony comprises multiple copies of the template nucleic acid molecule covalently attached in a linear sequence.
18 . The method of claim 1 , wherein nucleotides provided in the extending (c) comprise a mixture of labeled and unlabeled nucleotides.
19 . The method of claim 1 , wherein nucleotides provided in the extending (d) are non-terminated.
20 . The method of claim 10 , wherein flow steps of the plurality of first flow cycles further comprise (i) detection of a signal from a label of an incorporated labeled nucleotide and (ii) cleavage of the label.