Methods for nucleic acid analysis
Inventors
Almogy, Gilad • Oberstrass, Florian • BARAD, Omer • Shee, Chandan
Assignees
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Publication Number
US-12319959-B2
Publication Date
2025-06-03
Expiration Date
Abstract
The present disclosure provides methods and processes for increasing the efficiency and accuracy of nucleic acid sequencing using techniques such as polymerase chain reaction (PCR). The methods described herein can be used to achieve clonal amplification even with a greater than Poisson distribution of beads and/or nucleic acid templates into an emulsion. A PCR method may comprise generating a partition (e.g., a droplet) comprising at least two beads and/or at least two nucleic acid molecules and generating clonal amplification products corresponding to the nucleic acid molecule, at least a subset of which may be attached to a bead.
Core Innovation
The invention provides a method for nucleic acid processing in which a plurality of droplets are generated with a plurality of beads and a plurality of nucleic acid molecules. A droplet contains a bead comprising a first primer sequence and does not comprise any other beads, and each droplet further comprises at least two nucleic acid molecules with different nucleic acid sequences, where each nucleic acid molecule comprises a first adaptor sequence. The droplet further comprises one or more reagents including a second primer not attached to any beads, and the second primer has a first portion configured to hybridize to the first adaptor sequence and a second portion comprising a second primer sequence corresponding to the first primer sequence.
The second primer hybridizes to the first adaptor sequence of a first nucleic acid molecule, and the method generates one or more extension products of the first nucleic acid molecule comprising the second primer sequence or reverse complement thereof. An extension product is attached to the bead by annealing the second primer sequence or reverse complement thereof to the first primer sequence and extending the first primer, thereby generating amplification products of the first nucleic acid molecule attached to the bead and monoclonally amplifying the first nucleic acid molecule on the bead. The concentration ratio of the second primer within the droplet to the concentration of the first primer in the droplet is on the order of 10^-1 or less.
After generating amplification products, the method recovers the bead from the droplet and assays an amplification product attached to the bead to identify a sequence of the first nucleic acid molecule. The approach supports a droplet-based partitioning strategy using template-loading beads and sequencing-bead-associated primer functionality while maintaining monoclonality even when multiple templates are co-loaded. The disclosure also addresses Poisson distribution and double Poisson loading limitations using partitions with increased bead-to-template ratios to reduce template loss and polyclonality while enabling efficient reagent usage even at densities above Poisson expectations.
Claims Coverage
The document contains one independent claim. Across the dependent claims, the inventive features include a bead-bound first primer, a bead-free second primer at defined low concentration ratios, adaptor-hybridized extension and bead attachment, bead recovery and assay, droplet occupancy, sequencing with bead attachment, and droplet diameter constraints.
Bead-separated droplet composition with adaptor-containing nucleic acids and a bead-free second primer at low ratio
Generating a plurality of droplets with a plurality of beads and a plurality of nucleic acid molecules, where a droplet comprises a bead with a first primer sequence, at least two nucleic acid molecules having different nucleic acid sequences and each comprising a first adaptor sequence, and one or more reagents comprising a second primer not attached to any beads; the first primer sequence has no sequence complementarity with the first adaptor sequence.
Adaptor-hybridized extension followed by bead attachment and monoclonal amplification
With the second primer hybridized to the first adaptor sequence of the first nucleic acid molecule, generating extension products comprising the second primer sequence or reverse complement thereof, attaching an extension product to the bead by annealing the second primer sequence or reverse complement thereof to the first primer sequence and extending the first primer, and generating amplification products of the first nucleic acid molecule attached to the bead for monoclonal amplification.
Recover bead and assay bead-attached amplification products for sequence identification
Recovering the bead from the droplet and assaying an amplification product attached to the bead to identify a sequence of the first nucleic acid molecule.
Low second-to-first primer concentration ratio constraint
The ratio of the concentration of the second primer within a droplet to the concentration of the first primer in the droplet is on the order of 10^-2 or less.
Tight second-to-first primer concentration ratio constraint
The ratio of the concentration of the second primer within a droplet to the concentration of the first primer in the droplet is 10^-3 or less.
Droplet occupancy with bead and nucleic-acid template
At least 50% of the droplets contain at least one bead and at least one nucleic acid template.
Sequencing with amplification product remaining attached to a bead
Sequencing an amplification product while the amplification product remains attached to a bead.
Droplet diameter range constraint
A droplet having a diameter from about 1 nanometer to about 1 millimeter.
The core coverage is directed to droplet-based nucleic acid processing that uses adaptor hybridization with a bead-free second primer, bead attachment of extension products, monoclonal amplification on the bead, and recovery and bead-attached assay to identify the nucleic-acid sequence. The dependent claims add low second-to-first primer concentration ratios, droplet occupancy, sequencing with bead attachment, and droplet diameter range constraints.
Stated Advantages
Increase sequencing efficiency and accuracy by improving over double Poisson loading limitations through increased bead-to-template ratios that reduce template loss and polyclonality.
Enable efficient reagent usage even at densities above Poisson expectations.
Enables sequencing or read generation only from droplets that contain the intended bead and template combination, while other droplet subsets are cleared so they do not generate sequencing.
Maintains monoclonality or clonal amplification even when multiple templates are co-loaded in a partition.
Supports sequencing read architectures including paired-end read pairing using adaptor and primer correspondence.
Addresses rare inputs such as cfDNA and rare alleles.
Documented Applications
Nucleic-acid analysis using droplet or partition-based nucleic-acid processing with bead-attached clonal amplification products for assaying to identify nucleic-acid sequences.
Rare-input nucleic acid processing and sequencing, including cfDNA and rare alleles, as framed in the partial content.
Paired-end sequencing read architectures for identifying sequences of nucleic acids using bead-attached amplification products, as described in the partial content.
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