Methods and systems for processing polynucleotides
Inventors
Belgrader, Phillip • Bent, Zachary • Bharadwaj, Rajiv • Sreenivasa Gopalan, Vijay Kumar • Harada, Josephine • Hindson, Christopher • Rahimi Lenji, Mohammad • Lucero, Michael Ybarra • McDermott, Geoffrey • Meer, Elliott • Mikkelsen, Tarjei Sigurd • O'Keeffe, Christopher Joachim • Pfeiffer, Katherine • Price, Andrew D. • RYVKIN, PAUL • Saxonov, Serge • Stuelpnagel, John R. • Terry, Jessica Michele • Wheeler, Tobias Daniel • Wu, Indira • Ziraldo, Solongo Batjargal • Boutet, Stephane Claude • Taylor, Sarah • Srinivas, Niranjan
Assignees
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Publication Number
US-12534760-B2
Publication Date
2026-01-27
Expiration Date
Abstract
The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing and analyte characterization. Such polynucleotide processing may be useful for a variety of applications, including analyte characterization by polynucleotide sequencing. The compositions, methods, systems, and devices disclosed herein generally describe barcoded oligonucleotides, which can be bound to a bead, such as a gel bead, useful for characterizing one or more analytes including, for example, protein (e.g., cell surface or intracellular proteins), genomic DNA, and RNA (e.g., mRNA or CRISPR guide RNAs). Also described herein, are barcoded labelling agents and oligonucleotide molecules useful for “tagging” analytes for characterization.
Core Innovation
The invention provides compositions and partitioned assay formats that include barcode molecules in a partition, including droplets and wells. Barcode-bearing molecules are associated with analytes within individual partitions so that a first barcode molecule comprising a nucleic acid barcode sequence is hybridized to a first analyte that is not mRNA and is not hybridized via a poly-T sequence, where the first analyte comprises CRISPR crRNA or sgRNA. A second barcode molecule comprising the nucleic acid barcode sequence is hybridized to a second analyte that is mRNA and is hybridized via a poly-T sequence.
The invention relates to barcoding of cells and partitioning architectures for barcoded single-cell and multi-analyte assays that use aqueous droplets formed from microfluidic channel junctions and immiscible oil phases, including fluorinated oil with fluorosurfactants. The partition can be implemented as a plurality of droplets or as wells, and barcode molecules can be attached to a bead within the partition, including gel bead and degradable bead embodiments.
The barcode molecules can include a random N-mer sequence, and barcode molecules can include cell-identification sequences and partition-identification sequences. In related embodiments, a first plurality of barcode sequences hybridizes to CRISPR crRNA or sgRNA via a sequence that is not poly-T, while a second plurality hybridizes to mRNA via a poly-T sequence, with barcode sequences constant among the plurality of barcode molecules and random sequences varying among the plurality of barcode molecules.
Claims Coverage
The independent claims cover three related composition formats, centered on dual analyte recognition in a partition: mRNA hybridization via poly-T and CRISPR crRNA/sgRNA hybridization via non-poly-T sequences, with random N-mer or random sequence features and bead, droplet, or well implementations.
Differential poly-T and non-poly-T barcode hybridization to CRISPR and mRNA in a partition
A partition comprising a plurality of barcode molecules, including a first barcode molecule hybridized to a first analyte that is not mRNA and not hybridized via a poly-T sequence, where the first analyte comprises CRISPR crRNA or sgRNA, and a second barcode molecule hybridized to a second analyte that is mRNA and hybridized via a poly-T sequence.
CRISPR and mRNA dual hybridization in droplet partitions with bead-attached barcodes and random N-mer sequences
A plurality of droplets where a droplet comprises a gel bead having a plurality of barcode molecules attached thereto, including a first barcode molecule hybridized to a non-mRNA analyte comprising CRISPR crRNA or sgRNA without poly-T-mediated hybridization and a second barcode molecule hybridized to mRNA via a poly-T sequence, wherein the first barcode molecule and the second barcode molecule further comprise a random N-mer sequence.
First and second barcode populations for mRNA via poly-T and CRISPR analytes via non-poly-T with constant barcode sequences and varying random sequences
A partition comprising a bead having a plurality of barcode molecules attached thereto, where the plurality of barcode molecules comprises a first population of barcode molecules each hybridized to mRNA via poly-T sequences, and a second population of barcode molecules each hybridized to an analyte comprising CRISPR crRNA or sgRNA via a sequence that is not poly-T, where barcode sequences are constant among the plurality of barcode molecules and random sequences vary among the plurality of barcode molecules.
The claims collectively cover partitioned compositions that associate a constant nucleic acid barcode sequence with two different analyte classes by using poly-T mediated hybridization for mRNA and non-poly-T mediated hybridization for CRISPR crRNA/sgRNA. The coverage also includes droplet partitions with a gel bead and random N-mer, and bead-partition formats with two barcode populations having constant barcode sequences but varying random sequences.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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