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Publication Number
US-12522867-B2
Publication Date
2026-01-13
Expiration Date
Abstract
Described herein are methods for determining a sequence of a region of interest from an mRNA molecule. Sequenced polynucleotides can include a barcode region, a homopolymer region (e.g., a poly-A region), and a target region associated with the mRNA molecule. According to some methods, the barcode region omits the same base present in the homopolymer region. According to some methods, extension of the primer used for sequencing is stalled within the homopolymer region. According to some methods, sequencing flow cycles and the different barcode regions of the polynucleotides configured are such that the primer is extended to the end of the barcode region across the plurality of polynucleotides before being extended into the homopolymer region. According to some methods, two primers or a cleavable primer is used to separately sequence the barcode region and the target region.
Core Innovation
The invention provides a method of determining a sequence of a region of interest from an mRNA molecule by using mRNA-derived polynucleotides that include a barcode region, a homopolymer region, and a target region. The method forms a plurality of hybridized templates by hybridizing the polynucleotides with a primer, where the homopolymer region comprises a plurality of contiguous and identical bases and the target region comprises a sequence associated with the region of interest from the mRNA molecule.
A first phase determines the sequence of the barcode region by using labeled nucleotides and unlabeled nucleotides at a first proportion of labeled nucleotides to total nucleotides. A second phase extends the primer within the homopolymer region using labeled nucleotides complementary to the base present in the homopolymer region at a second proportion of labeled nucleotides to total nucleotides, with the second proportion greater than the first proportion, so that primer extension stalls within the homopolymer region.
After stalling, the method extends the primer to the end of the homopolymer region using unlabeled nucleotides complementary to the base present in the homopolymer region. Finally, the method determines the sequence of the target region using labeled nucleotides, thereby associating the region of interest sequence with the barcode while controlling primer extension behavior in the homopolymer region.
Claims Coverage
The partial content identifies one independent claim, with additional dependent claims refining the labeling scheme, homopolymer run-length constraints, region-of-interest definition, and quantitative labeling limits in specific steps. The inventive coverage centers on barcode sequencing with differential labeled/unlabeled nucleotide proportions, intentionally stalling primer extension within a homopolymer region, then using unlabeled nucleotides to complete extension through the homopolymer before sequencing the target region.
Barcode-target mRNA region determination with stalling through a homopolymer region
A method of determining a sequence of a region of interest from an mRNA molecule using hybridized templates formed from polynucleotides comprising a barcode region, a homopolymer region of contiguous identical bases, and a target region, where the barcode is sequenced, the primer extension stalls within the homopolymer region, extension resumes through the homopolymer, and the target region is sequenced using labeled nucleotides.
Differential labeled/unlabeled nucleotide proportions for barcode sequencing and stalled homopolymer extension
Determining the sequence of the barcode region using labeled nucleotides and unlabeled nucleotides at a first proportion of labeled nucleotides to total nucleotides, then extending the primer using labeled nucleotides complementary to the base present in the homopolymer region at a second proportion that is greater than the first proportion, such that primer extension stalls within the homopolymer region.
Unlabeled homopolymer completion followed by labeled target sequencing
Extending the primer to the end of the homopolymer region using unlabeled nucleotides complementary to the base present in the homopolymer region, and determining the sequence of the target region using labeled nucleotides after the homopolymer extension is completed.
Quantitative homopolymer run-length and labeled fraction constraints
Refining the method with constraints on the homopolymer region run length, including at least 8 contiguous and identical bases or at least 50 contiguous and identical bases, and constraining labeling during the homopolymer completion step by limiting that 0.1% or fewer of the nucleotides used in step (d) are labeled.
Region-of-interest definition and barcode association refinement
Defining the region of interest as either the 3′ untranslated region or a 5′ untranslated region of the mRNA molecule, and refining associations between the target region and unique barcode regions as part of the method’s determination workflow.
Across the independent claim and identified dependent refinements, the coverage is focused on sequencing a barcode and a target region from mRNA-derived polynucleotides while deliberately stalling primer extension within a homopolymer region using a higher proportion of labeled nucleotides, then completing extension through the homopolymer using unlabeled nucleotides and sequencing the target region with labeled nucleotides. Dependent claims further narrow run-length thresholds for the homopolymer region, labeling limits during homopolymer completion, and the specific mRNA region-of-interest definition.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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