Methods and systems for phasing sequencing strands and long-range sequencing

A method of synchronizing sequencing, 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, (i) providing a first plurality of nucleotides of a single base type, wherein at least a portion of the first plurality of nucleotides are labeled, and (ii) detecting a signal indicative of incorporation, or lack thereof, of a labeled nucleotide of the first plurality of nucleotides in the plurality of primers, until the signal falls below a first predetermined sequencing signal threshold;
• (d) extending, after the signal falls below the first predetermined sequencing signal threshold, 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.

A method of synchronizing sequencing primers within a sequencing cluster, comprising:

• hybridizing primers to polynucleotide copies within the sequencing cluster;
• extending the primers through a first region of the polynucleotide copies using nucleotides provided according to a first region flow order for a first predetermined number of sequencing flow steps, wherein, during each sequencing flow step in the first region flow order, a single type of nucleotide base is used and at least a portion of the nucleotides are labeled;
• extending, after the first predetermined number of sequencing flow steps, the primers through a second region of the polynucleotide copies using one or more re-phasing flow steps, wherein, during each of the one or more re-phasing flow steps, at least two different types of nucleotide bases are simultaneously used;
• and
• extending the primers through a third region of the polynucleotide copies using nucleotides provided according to a third region flow order comprising a plurality of sequencing flow steps, wherein, during each sequencing flow step in the third region flow order, a single type of nucleotide base is used and at least a portion of the nucleotides are labeled.

A method of synchronizing sequencing primers within a sequencing cluster, comprising:

• hybridizing primers to polynucleotide copies within the sequencing cluster;
• extending the primers through a first region of the polynucleotide copies using nucleotides provided according to a first region flow order comprising a plurality of sequencing flow steps until a sequencing signal falls below a first predetermined sequencing signal threshold, wherein, during each sequencing flow step in the first region flow order, a single type of nucleotide base is used and at least a portion of the nucleotides are labeled;
• extending, after the sequencing signal falls below the first predetermined sequencing signal threshold, the primers through a second region of the polynucleotide copies using one or more re-phasing flow steps, wherein, during each of the one or more re-phasing flow steps, at least two different types of nucleotide bases are simultaneously used;
• and
• extending the primers through a third region of the polynucleotide copies using nucleotides provided according to a third region flow order comprising a plurality of sequencing flow steps, wherein, during each sequencing flow step in the third region flow order, a single type of nucleotide base is used and at least a portion of the nucleotides are labeled.

A method of generating a long-range sequencing read, comprising:

• hybridizing primers to polynucleotide copies within a sequencing cluster;
• and
• alternatingly generating sequencing data and re-phrasing the primers across a plurality of sequencing regions and a plurality of re-phrasing regions, wherein:
• generating sequencing data for a sequencing region comprises extending the primers through the sequencing region using nucleotides provided according to a sequencing flow order comprising a plurality of sequencing flow steps, wherein, during each sequencing flow step in the sequencing flow order, a single type of nucleotide base is used and at least a portion of the nucleotides are labeled, and detecting the presence or absence of an incorporated labeled nucleotide while extending the primers through the sequencing region;
• and
• re-phrasing the primers comprises extending the primers through a re-phasing region using one or more re-phasing flow steps, wherein, during each of the one or more re-phasing flow steps, at least two different types of nucleotide bases are simultaneously used;
• wherein the distance between the start of a first sequencing region and the end of a final sequencing region is at least 300 bases in length.