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

Inventors

BARAD, Omer • Pratt, Mark • Trepagnier, Eliane • ETZIONI, YOAV • Oberstrass, Florian • Almogy, Gilad • Brinza, Dumitru

Assignees

Ultima Genomics Inc

Abstract

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.

Core Innovation

The invention synchronizes sequencing in a colony that includes a plurality of copies of nucleic acid molecules with sequence identity to a polynucleotide derived from a sample. Each nucleic acid molecule includes a first region, a second region, and a third region, and a plurality of primers is hybridized to the plurality of copies in the colony. Primer extension is divided across these regions while using different types of sequencing flows to control timing and alignment across the copies in the colony.

In the first region, primer extension is performed through a plurality of first flow cycles having a first predetermined number of flow steps, by providing a first plurality of nucleotides of a single base type in each flow step, wherein at least a portion of the first plurality of nucleotides is labeled. After the first predetermined number of flow steps, the method extends the primers through the second region by providing, in each flow step of a plurality of second flow cycles, a second plurality of nucleotides that comprises at least two base types. In the second region, one or more re-phasing flows with mixtures of at least two nucleotide base types are used to re-phase primers that become unsynchronized across the colony.

After extending through the second region, the method extends the 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. The document further describes triggering re-phasing based on a predetermined number of sequencing flow steps or based on measured sequencing signal or signal-to-noise ratio thresholds, and optionally repeating re-phasing across multiple region pairs to generate long-range sequencing reads spanning at least 300 bases, with reduced total phasing error and improved in-phase primer fractions, thereby reducing sequencing gaps.

Claims Coverage

The document includes one independent claim (clm-00001) directed to synchronizing sequencing in a colony, with the inventive features centered on dividing primer extension into first/second/third regions and using different flow-cycle nucleotide compositions, including a second region with at least two base types to re-phase primers.

Independent claim clm-00001 covers synchronizing colony sequencing by using labeled single-base-type flow cycles for a first region, a multi-base-type second region flow cycle set (at least two base types) positioned after a first predetermined number of flow steps to re-phase, and labeled single-base-type flow cycles for a third region.

Stated Advantages

Documented Applications