Methods and compositions for identifying or quantifying targets in a biological sample
Inventors
Stoeckius, Marlon • Smibert, Peter • Houck-Loomis, Brian
Assignees
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Publication Number
US-12071656-B2
Publication Date
2024-08-27
Expiration Date
Abstract
Compositions, kits and methods are described that comprise one or more constructs, each construct comprising a ligand attached or conjugated to a polymer construct, e.g., an oligonucleotide sequence, by a linker, each ligand binding specifically to a single target located in or on the surface of a cell. The polymer construct comprises a) an Amplification Handle; b) a Barcode that specifically identifies a single ligand; c) an optional Unique Molecular Identifier that is positioned adjacent to the Barcode on its 5′ or 3′ end; and d) an Anchor for hybridizing to a complementary sequence, e.g., for generation of a double-stranded oligonucleotide. These compositions are used in methods, including high throughput methods, for detecting one or more targets or epitopes in a biological sample. These compositions are also used in a high throughput method for characterizing a cell by simultaneous detection of one or more epitopes located in or on the cell and its transcriptome.
Core Innovation
The invention provides a method of simultaneously identifying from a single sequencing run a transcriptome and one or more proteins on one or more cells. Each cell originates from a biological sample and comprises one or more epitopes, where each epitope corresponds to one of the one or more proteins on the cells. Cells are used to generate complexes in which antibody or antibody fragments are conjugated to construct oligonucleotides.
The construct oligonucleotides include an amplification handle, an antibody barcode sequence, and an anchor sequence, and additional distinct constructs differ by the additional antibody or fragment and the additional antibody barcode sequence. Each complex is individually partitioned with a bead carrying capture oligonucleotides that hybridize to the anchor sequences and include a bead-specific barcode sequence unique to each bead. After lysis, the mRNA and construct oligonucleotides anneal to the capture oligonucleotides so that transcriptome and protein-associated nucleic acids become capture-associated in the same partition.
Separate libraries are generated and pooled for a single sequencing run, comprising a transcriptome amplification library with cDNAs derived from the annealed cellular mRNA and capture oligonucleotides, and a protein amplification library with DNAs derived from the annealed construct oligonucleotides and capture oligonucleotides. The method includes substantially separating by size the cDNA from the transcriptome amplification path and the DNA from the protein amplification path, and using antibody barcode sequences together with bead-specific barcode sequences to simultaneously identify the transcriptome and the one or more proteins on the one or more cells.
Claims Coverage
The consolidated claim coverage includes one independent method claim and dependent claims. It centers on antibody barcodes, bead partitioning with bead-specific barcodes, anchor-based hybridization, size separation to produce distinct transcriptome-versus-protein nucleic acid libraries, and parallel sequencing for simultaneous identification from the same cells.
Single-run simultaneous transcriptome and protein identification
A method simultaneously identifying from a single sequencing run a transcriptome and one or more proteins on one or more cells.
Antibody-oligonucleotide conjugates with amplification handle, antibody barcode, and anchor sequence
Generating complexes in which an antibody or fragment is conjugated to a construct oligonucleotide comprising an amplification handle, an antibody barcode sequence, and an anchor sequence, with additional distinct constructs differing by the additional antibody or fragment and the additional antibody barcode sequence.
Bead partitioning with capture oligonucleotides that hybridize to anchor sequences and bead-specific barcodes
Individually partitioning each complex with a bead conjugated to capture oligonucleotides that comprise sequences that hybridize to the anchor sequences and a bead-specific barcode sequence unique to each bead.
Annealing captured mRNA and constructs followed by dual cDNA/DNA generation
Lysing the cells to release cellular mRNA and construct oligonucleotides, annealing the mRNA and constructs to the capture oligonucleotides, and generating cDNA from the mRNA and DNA from the construct oligonucleotides.
Size-separated transcriptome and protein amplification libraries with pooled parallel sequencing
Substantially separating by size the cDNA and the DNA, generating independently from each other a transcriptome amplification library and a protein amplification library, pooling the two libraries, and sequencing them in parallel in a single sequencing run.
Dual barcode assignment for simultaneous identification
Using the antibody barcode sequences and the bead-specific barcode sequences to simultaneously identify the transcriptome and one or more proteins on the one or more cells.
cDNA/DNA separation using a 300-nucleotide fragment-length threshold
Substantially separating such that the cDNA comprises fragments longer than 300 nucleotides, and the DNA comprises fragments shorter than 300 nucleotides.
Multiplexing scale requiring at least 100 distinct additional constructs
Including one or more distinct additional constructs comprising at least 100 distinct additional constructs.
Cleavable covalent linker for construct conjugation
A linker having a cleavable covalent bond.
Disulfide cleavable covalent bond linker
The cleavable covalent bond is a disulfide bond.
PolyA anchor sequence with polyT capture oligonucleotides
The anchor sequence is a polyA sequence, and the capture oligonucleotides are polyT capture oligonucleotides.
The claims center on antibody-barcoded constructs with anchor sequences, bead partitioning with anchor-hybridizing capture oligonucleotides and bead-specific barcodes, annealing and parallel creation of transcriptome and protein libraries followed by size-based separation, and parallel sequencing in a single run with simultaneous assignment using antibody barcodes and bead barcodes. Dependent claims further specify the fragment-length threshold, multiplexing scale, linker cleavability including disulfide, and polyA/polyT anchor-capture pairing.
Stated Advantages
Simultaneous identification from a single sequencing run of a transcriptome and one or more proteins on one or more cells.
Simultaneous identification using antibody barcode sequences and bead-specific barcode sequences from parallel sequencing.
Generation of independently produced transcriptome and protein amplification libraries that are pooled and sequenced in parallel in a single sequencing run.
Documented Applications
Single-cell multimodal analysis of antibody-labeled epitopes together with transcriptome profiling using droplet microfluidics (CITE-seq).
Simultaneous, single-cell identification of cellular mRNA (transcriptome) and one or more proteins (cell-surface epitopes), with optional intracellular targets noted in the provided description.
Sample multiplexing using cell hashing/hashtagging.
Use/compatibility with flow cytometry benchmarks (as documented in the provided description).
Proof-of-principle species-mixing and PBMC profiling experiments showing concordance of RNA-derived species assignment with ADT protein marker assignment and improved immune phenotyping versus RNA-only.
Cell hashing using separate hashtag oligos for sample demultiplexing and multiplet/doublet identification, including genotyping/demuxlet comparison.
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