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Publication Number
US-12311332-B2
Publication Date
2025-05-27
Expiration Date
Abstract
Methods of generating a nucleic acid signature for identifying particles associated in a partition are provided. In one aspect, the method comprises: partitioning a sample into a plurality of partitions comprising a particle comprising a solid support surface, the solid support surface having a plurality of oligonucleotide primers conjugated thereon, wherein the oligonucleotide primers comprise a barcode sequence, and wherein the partitions have 0, 1, or more than 1 particles per partition; providing in a partition a substrate comprising a barcode sequence or repeating clonal barcode sequences; and in the partition, associating a first particle conjugated to oligonucleotide primers comprising a first barcode sequence and a second particle conjugated to oligonucleotide primers comprising a second barcode sequence to a barcode sequence from the substrate, thereby generating a nucleic acid signature for the particles in the partition.
Core Innovation
The invention describes a nucleic-acid signature method and corresponding partition library in which a sample is partitioned into multiple partitions such that partitions can comprise 0, 1, or more than 1 particle per partition. Particles are associated with oligonucleotide primers, and the primers carry barcode sequences, including cases where a same barcode sequence is present among a majority of primers on a particle. Each partition also comprises a substrate containing a barcode sequence or repeating clonal barcode sequences that can be released via a cleavable linker, including droplet-encapsulated substrate formats.
Released clonal barcode sequences from the substrate are associated with oligonucleotide primers on two or more particles in the same partition using association processes such as annealing and extension, or ligation. This association produces a unique combined nucleic-acid barcode signature that reflects which particles co-occur in a partition. The combined barcode signature is used for virtual linkage and deconvolution of pooled sequencing reads by linking signatures to co-occurring particles.
The partition library enables improved partition occupancy for partition-based assays, including increasing droplet occupancy while using more than one bead or particle per partition. Example implementations include clonal substrate formats such as hairpin molecules, and variants include linear and circular polynucleotide substrates, including circular or tandem-repeat formats with cleavable linkers. The document also describes partition library constructs and kits configured for downstream deconvoluting pooled sequencing data.
Claims Coverage
Independent claim clm-00001 is directed to a partition library having multiple partitions that include a cell with target nucleic acids, first and second particles conjugated to oligonucleotide primers with distinct barcode sequences, and clonal copies of at least one substrate barcode sequence or repeating clonal barcode sequence. The claim set includes dependent claims that refine the substrate or repeating barcode structure and add quantitative constraints such as average target nucleic acids per partition, minimum number of partitions, and minimum repeat counts.
Partition library with target nucleic acids in partitions
A partition library comprising two or more partitions, wherein at least some partitions comprise a cell comprising target nucleic acids.
Two particles with distinct primer barcode sequences per partition
At least some partitions further comprise a first particle conjugated to oligonucleotide primers comprising a first barcode sequence and a second particle conjugated to oligonucleotide primers comprising a second barcode sequence.
Clonal copies of substrate barcode sequences in partitions
At least some partitions further comprise clonal copies of at least one substrate barcode sequence or repeating clonal barcode sequence.
Average target nucleic acids per partition
The partition library is defined such that each partition contains, on average, between 0.1 and 5 target nucleic acids.
Minimum number of partitions
The partition library comprises at least 500 partitions.
Clonal copies associated with both first and second particles
Clonal copies of at least one substrate barcode sequence or repeating clonal barcode sequence are associated with both a first particle and a second particle.
Repeating clonal barcodes as tandem repeats separated by a cleavable linker
The repeating clonal barcode sequences are formed as tandem repeats separated by a cleavable linker.
Minimum repeat count in repeating clonal barcode sequences
The repeating clonal barcode sequences contain at least 10 repeats of the clonal barcode sequence.
Across the independent claim and its dependent refinements, the document claims a partition library with partitions containing a cell with target nucleic acids, first and second particles bearing distinct primer barcode sequences, and clonal copies of substrate barcode sequences, including repeating clonal barcode sequences. Refinements define quantitative constraints and structural parameters for repeating clonal barcodes.
Stated Advantages
Improved partition occupancy, including increasing droplet occupancy from about 10% to about 85–95% when using about 2–3 beads per partition.
Enables deconvoluting pooled sequencing reads using the unique combined nucleic-acid barcode signature derived from co-occurring particles in a partition.
Reduced dead volume.
Documented Applications
Partition-based assays that use nucleic-acid signatures to virtually link co-occurring particles and deconvolute pooled sequencing data.
Downstream analysis for deconvoluting pooled sequencing reads using combined nucleic-acid barcode signatures produced in partitions.
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