Accelerated sequencing methods
Inventors
Pratt, Mark • Almogy, Gilad • Brinza, Dumitru • Trepagnier, Eliane • BARAD, Omer • ETZIONI, YOAV • Oberstrass, Florian
Assignees
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Publication Number
US-12209278-B2
Publication Date
2025-01-28
Expiration Date
Abstract
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.
Core Innovation
The invention synchronizes sequencing primers within a sequencing cluster by hybridizing primers to polynucleotide copies in the sequencing cluster and extending the primers through a first region using a mixture of labeled and unlabeled nucleotides according to a first region flow cycle. The method generates sequencing data associated with a sequence of the first region by detecting the presence or absence of an incorporated labeled nucleotide.
The primers are then extended through a second region using one or more re-phasing flows without detecting incorporation of nucleotides into the primers during the one or more re-phasing flows. In each re-phasing flow, a mixture of at least two different types of nucleotide bases is used, and at least a portion of the nucleotides used in the second region are unlabeled.
The method further extends the primers through a third region using labeled nucleotides according to a third region flow cycle, where only a single type of nucleotide base is used at a time during a flow of the third region flow cycle. The re-phasing flows include mixtures in which three nucleotides are non-terminating and the fourth is a reversible terminator, and include multi-step flow sequences defined by inclusion or omission of base types.
Claims Coverage
The independent claim defines 9 inventive features centered on primer synchronization in a sequencing cluster with three regions and a non-detecting re-phasing second region. Dependent claims narrow the re-phasing flow mixtures, add detection in the first region, specify unlabeled nucleotides in the second region, and define sequencing-cluster formation by bridge amplification or emulsion PCR.
Synchronizing sequencing primers in a sequencing cluster
A method of synchronizing sequencing primers within a sequencing cluster by hybridizing primers to polynucleotide copies within a sequencing cluster.
First-region labeled/unlabeled extension according to a first region flow cycle
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.
Non-detecting re-phasing second-region extension with mixed base types
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.
Third-region labeled single-base-per-flow extension
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.
Re-phasing flows with reversible-terminator mixture compositions
The one or more re-phasing flows comprises multiple alternative flow mixtures where three nucleotides are non-terminating and the fourth is a reversible terminator, with the reversible terminator base type specified per flow.
Multi-step re-phasing flow sequence by inclusion/omission of base types
The one or more re-phasing flows comprises, in any order, four flow steps with specified nucleotide mixtures (A/C/G without T; T/C/G without A; T/A/G without C; T/A/C without G).
Detection of presence or absence of incorporated labeled nucleotide in the first region
The method generates 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.
Unlabeled nucleotides used in the second region
At least a portion of the nucleotides used to extend the primers through the second region are unlabeled nucleotides.
Sequencing cluster formed by bridge amplification or emulsion PCR
The sequencing cluster is formed by bridge amplification or emulsion PCR.
Overall claim coverage centers on using a first-region labeled/unlabeled extension, a second-region re-phasing extension that uses mixed base types but does not detect incorporation, and a third-region extension using labeled nucleotides with a single nucleotide base type per flow. Dependent coverage further specifies re-phasing flow compositions, detection behavior in the first region, unlabeled usage in the second region, and sequencing-cluster formation by bridge amplification or emulsion PCR.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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