US20180251825A1 - Methods and compositions for identifying or quantifying targets in a biological sample - Google Patents

Methods and compositions for identifying or quantifying targets in a biological sample Download PDF

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US20180251825A1
US20180251825A1 US15/887,144 US201815887144A US2018251825A1 US 20180251825 A1 US20180251825 A1 US 20180251825A1 US 201815887144 A US201815887144 A US 201815887144A US 2018251825 A1 US2018251825 A1 US 2018251825A1
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construct
oligonucleotide
barcode
sequence
anchor
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Marlon Stoeckius
Peter Smibert
Brian Houck-Loomis
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New York Genome Center Inc
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New York Genome Center Inc
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Publication of US20180251825A1 publication Critical patent/US20180251825A1/en
Priority to US17/245,479 priority patent/US20210371914A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • RNA-seq single cell RNA-seq
  • FACS/scRNA-seq approaches suffer from relatively low throughput and from an experimental bias in that only cell types chosen a priori are sorted and sequenced. Thus, these methods are not well suited for discovery of novel cell populations or for characterizing complex tissues that require the analysis of tens of thousands of cells.
  • a transition from plate-based approaches to the microfluidic/nanowell methods developed by Fluidigm and Wafergen allowed researchers to scale to enormous numbers of cells, alleviate the throughput bottleneck, bypass the experimental bias encountered by FACS and automate the cell capture and library preparation processes required for scRNA-seq.
  • Current droplet-based microfluidic platforms produce nanoliter-sized aqueous-in-oil emulsions at rates exceeding 1,000 droplets per second.
  • Microparticles with unique molecular Barcodes co-encapsulated with cells in droplets allow for grouping of transcripts originating from the same cell. This approach has significantly enhanced throughput by generating tens of thousands of individual single-cell reactions per experiment while achieving significant cost reductions associated with nanoliter volume reagent use.
  • droplet-based advances in single-cell genomics have dramatically changed the scale of scRNA-seq experiments, these methods suffer from a key disadvantage: All droplet-based single-cell RNA-sequencing methods lose important phenotypic information other than protein levels in general or cell-surface protein expression in particular (Table 1).
  • mRNA abundance is often a poor proxy for protein levels especially in developmental processes 4-6 .
  • the expression of cell-surface markers is traditionally measured via fluorescently-labeled antibodies by cell cytometry, and complex cell populations can be characterized by the combination of markers they express. For example, elaborate maps of cell types have been determined in recent years, based on protein markers in the immune and nervous systems 7 . This led to the use of cell cytometry as a diagnostic and monitoring tool in a number of disease areas, most prominently in oncology and immunology.
  • FACS-based approaches are limited in terms of the number of markers that can be assayed simultaneously and by the fact that cells chosen for analysis are biased by the selection of known surface markers.
  • compositions and methods are needed for qualitative and quantitative analysis of a multitude of cellular (and other) targets for diagnostic and research applications.
  • a composition comprises a construct that comprises a ligand attached or conjugated to a polymer construct, i.e., an oligonucleotide sequence, by a linker.
  • the ligand is designed to bind specifically to a target in a biological sample.
  • the polymer construct e.g., oligonucleotide sequence, comprises an Amplification Handle; a Barcode that specifically identifies the ligand, an optional Random Molecular Tag (RMT), or Unique Molecular Identifier (UMI), hereafter referred to as “UMI” that is positioned adjacent to the Barcode on its 5′ or 3′ end; and an Anchor for hybridization to a capture sequence that comprises a sequence complementary to the Anchor and for subsequent generation of double-stranded sequences.
  • RTT Random Molecular Tag
  • UMI Unique Molecular Identifier
  • the linker between ligant and polymer construct can be a cleavable covalent bond.
  • composition can further contain one or more “additional” constructs, which differ in at least one of target, ligand, and Barcode, as well as UMI from any other construct in the composition.
  • a composition comprises one or more “substantially identical” constructs.
  • each “substantially identical” construct differs from any other reference construct (e.g., the “first” construct or an “additional” construct) in the composition only in the identity of the sequence of the optional UMI or the absence of an UMI from the reference construct.
  • kits comprising one or more of the compositions and embodiments described herein, and optional reagents for performance of one or more methods.
  • a method for detecting one or more targets in a biological sample uses one or more of the compositions and constructs described herein.
  • the target is a cell surface antigen or epitope and the composition contains a single construct directed to that target, i.e., a “first” construct.
  • the composition contains multiple “substantially identical” constructs, i.e., substantially identical to the “first” construct, or one or more “additional” constructs directed to different targets and with consequently different components, as described above and defined below.
  • the method involves contacting a biological sample with one or more of the compositions described above. Additional steps involve washing to remove unbound constructs, and/or hybridizing each Anchor sequence in individual constructs to a capture sequence.
  • Another step involves extending the capture hybridized to the Anchor sequence to copy the construct Barcode, UMI and Amplification Handle onto double-stranded sequences.
  • the polymer construct Barcode sequences are thereafter amplified or detected to identify whether the biological sample expresses or contains a single target, one or more additional targets, or a combination of multiple targets.
  • the expression level of the targets in the sample are determined by detecting the amount of the corresponding polymer construct Barcodes normalized by an amount of any UMI or the mean amount of two or more UMIs in the treated sample.
  • a method as described above includes isolating individual cells, cell fragments, or populations of cells, from the biological sample bound to one or more of the constructs directed to detect one or more targets after the washing step. Still another step involves amplifying the double-stranded sequences with primers annealed to the Amplification Handles.
  • a method uses the compositions described herein for characterizing a cell by simultaneous detection of one or more epitopes located in or on the cell and/or its transcriptome.
  • One such method comprises contacting a biological sample containing cells with one or more of the compositions described herein.
  • the ligands are antibodies or fragments thereof that bind specifically to targeted epitopes located in a cell or on the surface of a cell.
  • Such a method can use the steps of the Drop-segs technique, e.g., encapsulating an individual single cell bound to one or more constructs into an aqueous droplet containing a microfluidics bead.
  • Each bead is conjugated to a capture oligonucleotide sequence.
  • mRNAs in the cell and the construct oligonucleotide sequence anneal to the polyT sequences of the capture oligonucleotide on the bead.
  • From the sequences annealed to the bead are generated double-stranded cDNAs containing the bead Barcode sequence and the reverse transcripts of the cellular mRNA and double-stranded DNA containing the bead Barcode sequence and the construct oligonucleotide sequence.
  • An amplification library containing the cDNA from the cell transcripts and the DNA containing the construct oligonucleotide sequence is generated.
  • the transcriptome of the library is associated with the cell identified by the antibody on a specifically identified construct simultaneously.
  • the polymer construct Barcode sequences are used to identify whether the single cell expresses the target epitope.
  • the transcriptome of the library is simultaneously associated with the cell identified as expressing the target.
  • constructs described above are used in a method of batch-barcoding or cell “hashtagging”.
  • An above-described construct e.g., an antibody or any ligand that binds to a cell, conjugated or associated with an oligonucleotide sequence comprising an Amplification Handle; a Barcode that specifically identifies the ligand, an optional Random Molecular Tag (RMT), or Unique Molecular Identifier (UMI), hereafter referred to as “UMI” that that is positioned adjacent to the Barcode on its 5′ or 3′ end; and an Anchor, e.g., polyA sequence) as described herein is used to label every cell within a sample prior to pooling.
  • RTT Random Molecular Tag
  • UMI Unique Molecular Identifier
  • a method for detecting a sample or target in a multiplex assay comprising: a) contacting a first sample with a first construct comprising a first ligand attached to a first oligonucleotide, wherein the first ligand binds specifically to a first target, and the first oligonucleotide comprises: i) a first amplification handle, ii) a first barcode that specifically identifies the first sample, and iii) a first anchor.
  • the method further comprises: b) contacting a second sample with a second construct comprising a second ligand attached to a second oligonucleotide, wherein the second ligand binds specifically to a second target, and the second oligonucleotide comprises: i) a second amplification handle, ii) a second barcode that specifically identifies the second sample, and iii) a second anchor.
  • the first target and the second target are the same target, and optionally, the first amplification handle and the second amplification handle are substantially identical, and optionally, the first anchor and the second anchor are substantially identical.
  • the method further comprises: c) contacting the first and the second samples with a third construct comprising a third ligand attached to a third oligonucleotide, wherein the third ligand binds specifically to a third target, and the third oligonucleotide comprises: (i) a third amplification handle, (ii) a third barcode that specifically identifies the third ligand, and (iii) a third anchor.
  • the method further comprises d) contacting the first and the second samples with a fourth construct comprising a fourth ligand attached to a fourth oligonucleotide, wherein the fourth ligand binds specifically to a fourth target, and the fourth oligonucleotide comprises: i) a fourth amplification handle, ii) a fourth barcode that specifically identifies the fourth ligand, and iii) a fourth anchor.
  • the third amplification handle and the fourth amplification handle are substantially identical, and are different from the first amplification handle and the second amplification handle.
  • the first anchor, the second anchor, the third anchor and the fourth anchor are substantially identical, and optionally comprise a polyA sequence of at least 10 nucleotides in length.
  • the third target and the fourth target are different targets, and optionally, the third target is different than the first or second targets, and optionally, the fourth target is different than the first or second targets.
  • the method further comprises e) contacting a third sample with a fifth construct comprising a fifth ligand that binds specifically to a fifth target, wherein the fifth target is optionally the same as the first target, and the fifth ligand is attached to a fifth oligonucleotide comprising: i) a fifth amplification handle, optionally substantially the same as the first amplification handle, ii) a fifth barcode that specifically identifies the third sample, and iii) a fifth anchor, optionally substantially the same as the first anchor, and optionally comprising a polyA sequence.
  • the method further comprises f) contacting the first and the second samples, and optionally additional samples with a sixth construct comprising a sixth ligand, wherein the sixth ligand binds specifically to a sixth target, and is attached to a sixth oligonucleotide comprising: i) a sixth amplification handle, optionally substantially the same as the third amplification handle, ii) a sixth barcode that specifically identifies the sixth target, and iii) a sixth anchor, optionally the same as the third anchor, and optionally comprising a polyA sequence.
  • the first and the second samples, an optionally one or more additional samples comprise one or more cells, and the first, second, third, fourth, fifth and sixth targets are present in, or on the surface of, at least one of the one or more cells.
  • the contacting of (a), (b), (c), (d), (e) or (f) comprises contacting the one or more cells of the first sample, the second sample, and optional additional samples with the first, second, third, fourth, fifth or sixth constructs.
  • the first and the second samples comprise one or more cell organelles, mitochondria, exosomes, liposomes, synthetic or naturally occurring vesicles, microvesicles, ectosomes, nuclei, bacteria, virus, beads, particles, microparticles, nanoparticles, macromolecules, and synthetic or naturally occurring lipid, phospholipid or membrane spheres
  • the first, second, third, fourth, fifth and sixth targets are present in, or on the surface of, at least one of the one or more cell organelles, mitochondria, exosomes, liposomes, synthetic or naturally occurring vesicles, microvesicles, ectosomes, nuclei, bacteria, virus, beads, particles, microparticles, nanoparticles, macromolecules, and synthetic or naturally occurring lipid, phospholipid or membrane spheres.
  • the contacting of (a), (b), (c), (d), (e) or (f) comprises contacting the one or more cell organelles, mitochondria, exosomes, liposomes, synthetic or naturally occurring vesicles, microvesicles, ectosomes, nuclei, bacteria, virus, beads, particles, microparticles, nanoparticles, macromolecules, and synthetic or naturally occurring lipid, phospholipid or membrane spheres of the first sample, the second sample, and optional additional samples with the first, second, third, fourth, fifth or sixth constructs.
  • the contacting of (a) and (b), and optionally (e) takes place prior to the contacting of any one of (c), (d) or (f).
  • the contacting of (c), (d) or (f) comprises contacting a mixture of the first sample, the second sample and optionally additional samples with the third, fourth or sixth constructs.
  • the first, second, third, fourth, fifth or sixth ligands comprise an antibody, or antigen binding fragment thereof.
  • the first, second, third, fourth, fifth or sixth anchor is located 3′ of the first, second, third, fourth, fifth or sixth amplification handle, respectively, and 3′ of the first, second, third, fourth, fifth or sixth barcode, respectively; and optionally, (ii) the first, second, third, fourth, fifth or sixth amplification handle is located 5′ of the first, second, third, fourth, fifth or sixth barcode, respectively, and 5′ of the first, second, third, fourth, fifth or sixth anchor, respectively.
  • the method further comprises washing the first sample, the second sample, or a mixture of the first sample and the second sample, and optionally additional samples after any one or more of steps (a), (b), (c), (d), (e), or (f) to remove unbound constructs.
  • a first droplet comprising a first bead conjugated to a plurality of a first capture oligonucleotide comprising, from 5′ to 3′, a seventh amplification handle, a seventh barcode identifying the first bead, and a sequence complementary to the first, second, third, fourth, fifth or sixth Anchor sequence
  • the method further comprises lysing the first and second single cells thereby providing a first lysate encapsulated in the first droplet and a second lysate encapsulated in the second droplet, wherein the first and second lysates optionally comprise mRNA.
  • the method further comprises contacting the lysate of the first and second cells with a polymerase.
  • the method further comprises generating cDNA and double stranded oligonucleotide sequences of the first, second, third, fourth, fifth or sixth oligonucleotides.
  • a method for detecting one or more targets in a biological sample comprising contacting the biological sample with one or more of: a) a composition comprising a first construct that comprises a first ligand attached or conjugated to a polymer construct by a linker, said first ligand binding specifically to a first target, and said polymer construct comprising: an Amplification Handle; a Barcode that specifically identifies said first ligand; an optional Unique Molecular Identifier that is positioned adjacent to the Barcode on its 5′ or 3′ end; and an Anchor for hybridizing to a capture sequence that comprises a sequence complementary to said Anchor; b) a composition comprising at least one additional construct, which construct comprises an additional ligand attached or conjugated to an additional polymer construct by a linker, said additional ligand binding specifically to an additional target, and said additional polymer construct comprising an Amplification Handle; an additional Barcode that specifically identifies said additional ligand; an optional additional Unique Molecular Identifier that is
  • a high-throughput method for detecting one or more epitopes in a biological sample comprising contacting a biological sample with one or more of (i) a composition comprising a first construct that comprises a first antibody or fragment thereof that binds specifically to a first epitope, said first antibody or fragment attached or conjugated to a first polymer construct by a linker, wherein the first polymer construct comprises: an Amplification Handle; a Barcode Sequence that specifically identifies said first antibody or fragment from any other antibody or fragment that recognizes a different epitope, an optional Unique Molecular Identifier sequence that is positioned adjacent to the 5′ or 3′ end of the Barcode, and an Anchor sequence for hybridizing to a capture sequence that comprises a sequence complementary to said Anchor; (ii) a composition of (i) comprising at least one additional construct, which comprises an additional antibody or fragment thereof attached or conjugated to an additional polymer construct by a linker, said additional antibody or fragment thereof binding specifically to an additional epitope
  • a method for detecting at least two targets in at least a first and a second sample comprising: a) contacting the first sample with a first construct comprising a first ligand attached to a first oligonucleotide, wherein the first ligand binds specifically to a first target, and the first oligonucleotide comprises: i) a first amplification handle, ii) a first barcode that specifically identifies the first sample, and iii) an anchor comprising a polyA sequence; b) contacting the second sample with a second construct comprising the first ligand attached to a second oligonucleotide, wherein the second oligonucleotide comprises: i) the first amplification handle, ii) a second barcode that specifically identifies the second sample, and iii) the anchor; c) contacting the first and the second samples with a third construct comprising a second ligand attached
  • kits comprising: a) a first construct comprising a first ligand attached to a first oligonucleotide, wherein the first ligand binds specifically to a first target, and the first oligonucleotide comprises: i) a first amplification handle, ii) a first unique barcode configured to specifically identify a first sample, and iii) an anchor comprising a polyA sequence; b) a second construct comprising the first ligand attached to a second oligonucleotide, wherein the second oligonucleotide comprises: i) the first amplification handle, ii) a second unique barcode configured to specifically identify a second sample, and iii) the anchor; c) a third construct comprising a second ligand attached to a third oligonucleotide, wherein the second ligand binds specifically to a second target, and the third oligonucleot
  • composition comprising a construct comprising a ligand attached to an oligonucleotide, wherein the ligand binds specifically to a target, and the oligonucleotide comprises: i) an amplification handle, ii) a unique barcode configured to specifically identify a first sample, and iii) an anchor, optionally comprising a polyA sequence.
  • the methods described herein are high throughput methods and employ other known detection and sequencing techniques.
  • FIGS. 1A through 1C are schematic graphs showing the CITE-seq process and components enable simultaneous detection of single-cell transcriptomes and protein markers.
  • FIG. 1A is an illustration showing an embodiment of a construct described herein in which antibodies (ligands) are linked to a polymer construct, which in this embodiment is an oligonucleotide sequence a disulfide bridge (linker) and containing functional sequence components (Amplification Handle and PCR handle) and a unique antibody identifier Barcode followed by a polyA tail (Anchor).
  • a polymer construct which in this embodiment is an oligonucleotide sequence a disulfide bridge (linker) and containing functional sequence components (Amplification Handle and PCR handle) and a unique antibody identifier Barcode followed by a polyA tail (Anchor).
  • FIG. 1B is an illustration showing that Drop-seq beads are microparticles containing the polymer construct oligonucleotide sequence(s) with the functional features of Amplification Handle (PCR handle), a unique cell Barcode, followed by a unique molecular identifier (UMI) and a polyT tail (Anchor).
  • PCR handle Amplification Handle
  • UMI unique molecular identifier
  • Anchor a polyT tail
  • FIG. 1C is a schematic diagram of an embodiment of the CITE-seq protocol.
  • Cells are incubated with antibodies ( 1 ), washed ( 2 ) and passed through a microfluidic chip where a single cell and one bead is encapsulated in a droplet ( 3 ) and ( 4 ).
  • cell lysis 5
  • mRNAs and antibody-oligonucleotide constructs bind to Drop-seq beads ( 6 ).
  • Reverse transcription and template switch is performed in bulk after droplet emulsion breakage ( 7 ).
  • full length cDNA ( 8 a ) and antibody-oligo construct products ( 8 b ) can be separated by size and amplified independently.
  • FIG. 1D is an illustration showing an embodiment of a construct described herein in which antibodies (ligands) are linked to a polymer construct containing functional sequence components (Amplification Handle and PCR handle) and a unique antibody identifier Barcode followed by a polyA tail (Anchor).
  • FIGS. 2A through 2E are graphs showing that CITE-seq accurately identifies different species in mixing experiment.
  • FIG. 2A is a gel electrophoresis result as well as illustrations of the detected molecules.
  • Antibody-oligo complexes ( 1 ) appear as high molecular weight smear on agarose gel and can be cleaved by reducing the disulfide bond ( 2 ).
  • FIG. 2B are graphs illustrating two antibody-oligos.
  • Anti-Mouse Integrin Beta-1 (CD29) antibodies are linked to Barcoded oligo 1 containing a disulfide bridge linker, an Amplification Handle (also referred to as common sequence or PCR handle) , a unique antibody identifier Barcode (5′-ATGTCCT-3′) and a UMI containing 4 nt followed by a polyA tail (top panel).
  • Anti-human CD29 antibodies are linked to Barcoded oligo 2 containing a disulfide bridge, a common sequence (Amplification Handle, PCR handle) , a unique antibody identifier Barcode (5′-GCCATTA-3′) and a UMI containing 4 nt followed by a polyA tail (bottom panel).
  • FIG. 2C are results of gel electrophoresis and capillary electrophoresis trace of the full length cDNAs and oligos derived from the antibody-oligos. After reverse transcription and SMART PCR, two distinct product populations can be observed (right panel). These can be size separated into full length cDNAs (top panel, capillary electrophoresis trace) and antibody-oligo product (bottom panel) and amplified independently.
  • FIG. 2D is a dot plot showing the readout from RNA-seq as well as mouse and human antibody specific oligo sequences obtained in the same sequencing run.
  • Human and mouse cells were incubated with oligo-tagged-antibodies specific for human or mouse cell surface markers (integrin beta, CD29). Cells were then passed through the Drop-seq workflow at higher concentration to allow for multiple cell encapsulation. Species in each droplet (dots on scatterplot) were then determined by mRNA sequencing (human RNA: circled by a solid line except for a small number of outliers; mouse RNA: circled by a dashed line except for a small number of outliers; mixed species RNA: rest of the dots except some outliers mentioned above).
  • FIG. 2E is a dot plot showing the primary classification of counted cells by sequencing mRNA and cDNAs generated therefrom. Dots represented human cells and mouse cells are labelled by a solid circle and a dashed circle respectively.
  • FIG. 3A is a plot displaying the results of a CITE-seq analysis of 8,700 mononuclear blood cells labeled with 10 CITE-seq antibody constructs as described herein having the components as set out in Table 2 (See Example 7 below).
  • tSNE t-distributed Stochastic Neighbor Embedding
  • clustering are performed using canonical correlation analysis, which integrates protein and RNA measurements.
  • FIG. 3B is a CITE-seq analysis of the same dataset of FIG. 3A using RNA data alone.
  • the symbols in the figure are Mono (for monocytes), B for B cells, T for T cells, NK for natural killer cells, DC for conventional dendritic cells, pDC for plasmacytoid DC, Pre for precursors, and Ery for erythroblasts. Comparing FIG. 3A and FIG. 3B demonstrates enhanced resolution when using multi-modal data.
  • FIG. 3C shows bi-axial plots of CITE-seq antibody data for select antibodies, i.e. Construct Nos. 1, 3, 4, 6, 7, and 9 of Table 2 (See Example 7). These data show that, in contrast to information obtained by flow cytometry, using CITE-seq methodology and compositions makes available the transcriptome for every single cell (every dot) within the plot. Cells can therefore be further analyzed and classified based on their RNA data, protein data, or both.
  • FIG. 4 is a series of bi-axial plots generated by multiplexing 8,700 mononuclear blood cells labeled with the 10 antibody constructs of Table 2 (Example 7) in a CITE-seq analysis as described herein. Shown are bi-axial plots of CITE-seq antibody data for all 10 antibodies. These data are comparable to the information obtained by flow cytometry, using CITE-seq methodology and compositions also makes available the transcriptome for every single cell (every dot) within the plot. Cells can therefore be further analyzed and classified based on their RNA data, protein data, or both.
  • FIG. 5A is a plot showing the RNA clustering of about 4,000 peripheral blood mononuclear cells (PBMC), containing B cells, NK cells, mouse cells, Natural Killer T cells, Monocytes, CD16 Monocytes, CD4 cells and CD8 cells.
  • PBMC peripheral blood mononuclear cells
  • tSNE t-distributed Stochastic Neighbor Embedding
  • FIG. 5B shows 6 histogram profiles of CLR (centered log ratio) -transformed ADT (antibody derived tag) levels in clusters of B cells, NK cells, mouse cells, Natural Killer T cells, Monocytes, CD16 Monocytes, CD4 cells and CD8 cells exposed to compositions as described in Table 3.
  • CLR center log ratio
  • ADT antibody derived tag
  • compositions comprises a Ligand which is either an anti-CD3 antibody, anti-CD4 antibody or anti-CD8 antibody, covalently (directly) linked to a polymer construct, in this example a DNA oligonucleotide, containing an Amplification Handle compatible with Illumina Truseq Small RNA, a 10 nucleotide Barcode that is unique to each antibody to identify the Ligand, and a 30 nucleotide polyA tail Anchor for hybridizing to a capture sequence that comprises a sequence complementary to the Anchor.
  • Other such compositions comprises a Ligand which is either an anti-CD3 antibody, anti-CD4 antibody or anti-CD8 antibody, linked via a streptavidin-biotin linkage (SAV) as used in proof of principle experiments ( FIGS.
  • SAV streptavidin-biotin linkage
  • a polymer in this example a DNA oligonucleotide, containing an Amplification Handle compatible with Illumina Truseq Small RNA, a 10 nucleotide Barcode that unique to each antibody to identify the Ligand, and a 30 nucleotide polyA tail Anchor for hybridizing to a capture sequence that comprises a sequence complementary to the Anchor.
  • the histogram profiles in the different populations e.g., NK cells, CD4, CD8 are comparable between the SAV and direct conjugation.
  • FIGS. 6A-6F show sample multiplexing using DNA-barcoded antibodies.
  • FIG. 6A is a schematic overview of sample multiplexing by cell hashing.
  • Cells from different samples are incubated with DNA-barcoded antibodies recognizing ubiquitous cell surface proteins.
  • Distinct barcodes referred to as ‘hashtag’-oligos, HTO
  • HTO Distinct barcodes
  • FIG. 6B is a representative scatter plot showing raw counts for HTO A and HTO B, across all cell barcodes. Both axes are clipped at 99.9% quantiles to exclude visual outliers.
  • FIG. 6C is a heatmap of all normalized and scaled HTO levels, based on our classifications. Doublets and multiplets express more than one HTO. Negative populations contain HEK-293T and mouse NIH-3T3 cells that were spiked into the experiments as negative controls. Cells with multiple “hashtag” signals are likely doublets, and the frequency of these cells matches with expected multiplet rates for the assay described in Example 10.
  • FIG. 6D shows tSNE embedding of the HTO dataset. Cells are colored and labeled based on our classifications. Eight singlet clusters and all 28 cross-sample doublet clusters are clearly present.
  • FIG. 6E shows a distribution of RNA UMIs per cell barcode in cells that were characterized as singlets (red), doublets (violet) or negative (grey).
  • FIG. 6F shows a transcriptome-based clustering of single-cell expression profiles reveals distinct immune cell populations interspersed across donors.
  • B B cells
  • T T cells
  • NK natural killer cells
  • mono monocytes
  • DC dendritic cells
  • pDC plasmacytoid dendritic cells
  • Plasma cells Cells are colored based on their HTO classification (donor ID), as in FIG. 6D .
  • FIGS. 7A-7E show the validation of cell “hashing” using demuxlet.
  • FIG. 7A shows a row-normalized “confusion matrix” comparing demuxlet and HTO classifications. Each value on the diagonal represents the fraction of barcodes for a given HTO classification that received an identical classification from demuxlet.
  • FIG. 7B is a count distribution of the most highly expressed HTO for groups of concordant and discordant singlets. Both groups have identical classification strength based on cell “hashing”.
  • FIG. 7C shows that discordant singlets have lower UMI counts, suggesting that a lack of sequencing depth contributed to ‘ambiguous’ calls from demuxlet.
  • FIG. 7D are RNA UMI distributions for discordant and concordant multiplets. Only concordant multiplets exhibit increased molecular complexity, suggesting that both methods are conservatively overcalling multiplets in discordant cases.
  • FIG. 7E shows that demuxlet assigns lower multiplets posterior probabilities to discordant calls.
  • FIGS. 8A-8F show that cell “hashing” enables efficient experimental optimization and identification of low-quality cells.
  • FIGS. 8A to 8C are graphs showing the results of the performance of a titration series to assess optimal staining concentrations for a panel of CITE-seq immunophenotyping antibodies. Normalized ADT counts for CD8 ( FIG. 8A ) CD45RA ( FIG. 8B ) and CD4 ( FIG. 8C ) are depicted for the different concentrations used per test.
  • FIG. 8D shows a titration curve, depicting the staining index (SI) for these three antibodies across the titration series.
  • SI staining index
  • FIG. 8E shows that cells with low UMI counts can be distinguished from ambient RNA using HTO classifications. Classified singlets group into canonical hematopoietic populations.
  • FIG. 8F shows barcodes classified as “negative” do not group into clusters, and likely represent ‘empty’ droplets containing only ambient RNA.
  • compositions described herein increase the sensitivity of a variety of assay methodologies. Use of the compositions and methods to detect multiple targets in a complex environment is highly scalable and only limited by the number of specific ligands, e.g., antibodies, that are available, as opposed to fluorescent assay methods that are limited by spectral overlap of available fluorophores. For instance, flow cytometry allows the routine measurement of up to 15 parameters per ce11 17,18 .
  • the compositions described herein which employ molecular barcoding of ligands (e.g., antibodies) allow multiplexing to virtually any number and should even outcompete mass cytometry-based parallelization (CyTOF up to 100 tags) 18 .
  • compositions and methods described in detail below allows for simultaneous measurement of large numbers of established antibody-based markers along with unbiased single-cell transcriptome data, on the scale of tens of thousands of cells per experiment.
  • This technique Cellular Indexing of Transcriptome and Epitopes by sequencing (CITE-seq), using the compositions described herein.
  • CITE-seq Cellular Indexing of Transcriptome and Epitopes by sequencing
  • other techniques may use the described compositions to enhance the study and understanding of cell types and cell populations, such as cataloging cell types in healthy individuals or studying post-transcriptional gene regulation in development and disease.
  • the efficiency of any number of diagnostic techniques and applications for assaying various disease states can be enhanced by use of the compositions described herein.
  • the methods and compositions described herein greatly expand the power of single-cell phenotyping by combining information from both proteins and transcripts from the same single cells at an unprecedented scale.
  • the term “construct” refers to a chemically synthesized or genetically engineered assemblage that comprises a ligand attached (covalently, non-covalently, or otherwise as noted herein) to at least one polymer construct (e.g., in one embodiment, an oligonucleotide sequence) by a linker.
  • Each polymer construct comprises several functional elements: an Amplification Handle; a Barcode that specifically identifies the attached ligand, an optional Unique Molecular Identifier that is positioned adjacent to the Barcode on its 5′ or 3′ end, and an Anchor for hybridizing to a capture sequence that comprises a sequence complementary to the Anchor.
  • a construct comprises a single ligand linked to multiple identical polymer constructs.
  • each polymer construct is directly linked to the ligand (one linkage per polymer construct).
  • the polymer constructs are linked to the ligand as concatamers (multiple polymer constructs per single ligand linkage). For example, a single ligand (i.e., a monoclonal antibody) may be linked to from 1 to 50 polymer constructs.
  • a single strand of a nucleic often comprises a 5′ (5-prime) end and a 3′ (3-prime) end.
  • the terms 5′ and 3′ therefore refer to a relative position on a single strand of a nucleic acid.
  • the relative position of certain elements or sequences of a nucleic acid e.g., a handle, a barcode and an achor
  • a nucleic acid may include, from 5′ to 3′, a handle, a barcode and an anchor and may be represented as: 5′-handle- barcode-anchor-3′.
  • the barcode and the anchor may be referred to as being 3′ of the handle.
  • the handle and the barcode may be referred to as being 5′ of the anchor.
  • the position of the handle in the above example may also be referred to as adjacent to the barcode.
  • the barcode may be referred to as flanked by the handle and the anchor. Accordingly, one of skill in the art would know what is meant by the positional terms 3′ and 5′.
  • Such positional language, as used herein, unless explicitly indicated otherwise, does not imply that additional nucleic acid sequences are not interposed between the reference elements.
  • additional sequences e.g., a UMI
  • polymer refers to any backbone of multiple monomeric components that can function to bind to the selected ligand and/or Anchor component and be utilized in a downstream assay.
  • This assay may utilize the activity of one or more enzymes, for example reverse transcriptases, DNA or RNA polymerases, DNA or RNA ligases, etc.
  • Such polymers or monomeric components include oligonucleotides (e.g., DNA, RNA, synthetic or recombinant DNA or RNA bases or analogs of DNA or RNA bases), peptide nucleic acids (i.e., a synthetic nucleic acid analog in which natural nucleotide bases are linked to a peptide-like backbone instead of the sugar-phosphate backbone found in DNA and RNA), locked nucleic acids (LNA; see, e.g., Grunweller A and Hartmann RK, “Locked nucleic acid oligonucleotides: the next generation of antisense agents?” BioDrugs 2007. 21(4):235-43), or polyamide polymers (see, e.g.
  • a polymer construct or a functional component thereof may also be exemplified as a specific polymer or monomeric component, such as an oligonucleotide sequence, a nucleic acid, a nucleic acid sequence, etc.
  • oligonucleotide “nucleic acid” or nucleotide” or a similar specific example of a monomer or polymer is used in this specification, it should also be understood to mean that the polymer construct or component may be formed of any suitable polymer as described in this paragraph.
  • first “additional” and “substantially identical” are used throughout this specification as reference terms to distinguish between various forms and components of constructs.
  • a “first construct” may define a construct with certain specified components in which a single specified “first” ligand binds a specific “first” target.
  • the “first” Barcode is specific for the first ligand; the UMI identifies only that “first” polymer construct, and the Anchor binds a specified complementary sequence.
  • additional construct refers to a construct (e.g., a second, third or fourth construct) that differs from any other construct used in the compositions and methods defined herein in the identity of the target, ligand, and Barcode.
  • an additional construct differs from other constructs in the compositions or methods by the identity of target, ligand, Barcode, UMI and Anchor.
  • Each additional construct comprises an additional ligand attached or conjugated to an additional polymer construct by a linker.
  • the additional ligand binds specifically to an additional target different from that of the first target.
  • the linker between the ligand and the additional polymer construct may be the same or different from the linker in the first construct.
  • the additional polymer construct also differs in the identity of its functional elements.
  • the Amplification Handle may be the same or different from that used in the first construct.
  • the additional Barcode that specifically identifies the additional ligand does not identify any other ligand.
  • the optional additional UMI that is positioned adjacent to the additional Barcode on its 5′ or 3′ end, is specific for the additional polymer construct.
  • the additional Anchor has the same or a different sequence for hybridizing to the same or a different capture complementary sequence than that to which the first Anchor binds.
  • each “additional” ligand, “additional” target, “additional” Barcode and “additional” UMI components of each additional construct differs from the corresponding component in any other construct in the described composition or method.
  • first”, “second”, “third”, “fourth”, “fifth”, “sixth, “seventh” and “eighth”, refer to an element of the invention (e.g., construct, ligand, barcode, oligonucleotide, capture oligonucleotide, bead, target, anchor, amplification handle, and the like), where the recited “first”, “second”, “third”, “fourth”, “fifth”, “sixth, “seventh” and “eighth” elements may be the same or may be different.
  • binds refers to a ligand that binds to an indicated target in preference to binding to other targets (e.g., other molecules, other peptides, or other antigens) as determined by, for example, a suitable in vitro assay (e.g., an Elisa, Immunoblot, Flow cytometry, and the like).
  • targets e.g., other molecules, other peptides, or other antigens
  • a suitable in vitro assay e.g., an Elisa, Immunoblot, Flow cytometry, and the like.
  • a ligand that binds specifically to a target displays a specific binding interaction with the target that discriminates over non-specific binding interactions with other targets (e.g., any other protein, antigen, molecule, etc.) by about 2-fold or more, often about 10-fold or more, and sometimes about 100-fold or more, 1000-fold or more, 10,000- fold or more, 100,000-fold or more, or 1,000,000-fold or more.
  • targets e.g., any other protein, antigen, molecule, etc.
  • two or more nucleic acids are substantially identical.
  • Two or more nucleic acids, or portions thereof that are substantially identical refers to a nucleotide sequences of the two or more nucleic acids (e.g., two or more oligonucleotides, capture oligonucleotides, anchors, amplification handles, barcodes, and the like) that share at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% percent identity.
  • percent identical refers to sequence identity between two amino acid sequences. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same nucleotide, then the nucleic acid sequences are identical at that position. Expression as a percentage of identity refers to a function of the number of identical nucleotides, or a derivative or variant thereof, at corresponding positions (e.g., as defined by an alignment) shared by the compared sequences. Various alignment algorithms and/or programs may be used to determine percent identity, non-limiting examples of which include FASTA, BLAST, or ENTREZ.
  • FASTA and BLAST are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default settings.
  • ENTREZ is available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD.
  • substantially identical construct refers to a number of constructs or components, which differ from a reference construct, e.g., the “first” construct or a specific additional construct, only in the sequence of an optional Unique Molecular Identifier or its absence from the construct.
  • each one of a substantially identical construct shares the same target, ligand, Amplification Handle, Barcode and Anchor as does the reference (first or additional) construct.
  • each one of the substantially identical constructs shares the same target, ligand, Barcode and Anchor as does the reference (first or additional) construct.
  • a substantially identical construct to the “first construct” differs from the reference “first” construct in the sequence and/or presence of the UMI.
  • the substantially identical additional construct differs from the reference additional construct in the UMI and the Amplification Handle.
  • attachment or “attach” as used herein to describe the interaction between the components of the constructs is meant covalent attachments or a variety of non-covalent types of attachment.
  • Other attachment chemistries useful in assembling the constructs described herein include, but are not limited to, thiol-maleimide, thiol-haloacetate, amine-NHS, amine-isothiocyanate, azide-alkyne (CuAAC), tetrazole-cyclooctene (iEDDA) (See, e.g., reference 24 and other references therein).
  • each polymer construct is linked to the ligand by an irreversible covalent link.
  • each polymer construct is linked to the ligand by a cleavable covalent link, for example a disulfide link or a photocleavable linker.
  • target refers to any naturally occurring or synthetic biological or chemical molecule.
  • the target refers to any biological or chemical molecule expressed on the surface of a cell.
  • a target refers to any biological or chemical molecule on the surface of, or within an exosome, a nucleus, a cellular organelle, a virus or a bacteria.
  • a target is a cell-surface protein.
  • a target is a cell.
  • a target is a nucleus, exosome, bacteria or phage.
  • the target refers to any biological or chemical molecule expressed intracellularly.
  • the target refers to any biological or chemical molecule occurring in a naturally occurring, synthetic, recombinantly engineered or isolated library, panel, or mixture of targets. In another embodiment, the target refers to any biological or chemical molecule occurring in a biological sample.
  • first target and each “additional target” (e.g., a second, third, fourth target, or the like) refer to different targets.
  • the first and additional targets may independently be selected from a peptide, a protein, an antibody or antibody fragment, an affibody, a ribonucleic acid sequence or deoxyribonucleic acid sequence, an aptamer, a lipid, a polysaccharide, a lectin, or a chimeric molecule formed of multiples of the same or different targets.
  • the targets are cell surface antigens or epitopes.
  • a sample is a biological sample.
  • a “biological sample” as used in the methods described herein refers to a naturally-occurring sample or deliberately designed or synthesized sample or library containing one or more selected targets.
  • a sample contains a population of cells or cell fragments, including without limitation cell membrane components, exosomes, and sub-cellular components.
  • the cells may be a homogenous population of cells, such as isolated cells of a particular type, or a mixture of different cell types, such as from a biological fluid or tissue of a human or mammalian or other species subject.
  • Still other samples for use in the methods and with the compositions include, without limitation, blood samples, including serum, plasma, whole blood, and peripheral blood, saliva, urine, vaginal or cervical secretions, amniotic fluid, placental fluid, cerebrospinal fluid, or serous fluids, mucosal secretions (e.g., buccal, vaginal or rectal).
  • Still other samples include a blood-derived or biopsy-derived biological sample of tissue or a cell lysate (i.e., a mixture derived from tissue and/or cells). Other suitable tissue includes hair, fingernails and the like.
  • Still other samples include libraries of antibodies, antibody fragments and antibody mimetics like affibodies.
  • samples may further be diluted with saline, buffer or a physiologically acceptable diluent. Alternatively, such samples are concentrated by conventional means. Still other samples can be synthesized or engineered collections of chemical molecules, proteins, antibodies or any other of the targets described herein.
  • a sample is often obtained from, or derived from a specific source, subject or patient. In some embodiments, a sample is often obtained from, derived from, or associated with a specific experiment, lot, run or repetition. Accordingly, in certain embodiments, each of a plurality of samples (e.g., samples derived from different sources, different subjects, or different runs, for example) can be identified and/or differentiated using a method or composition described herein.
  • a sample is detected, tracked, tagged and/or identified by a method of hashtagging described herein.
  • the presence, amount or absence of a sample is determined by a method of hashtagging described herein.
  • a target e.g., a cell, nucleus, protein, etc.
  • a target that is derived from a specific sample, or source, is detected, tracked, tagged and/or identified by a method of hashtagging described herein.
  • a sample comprises one or more organelles, mitochondria, exosomes, liposomes, synthetic or naturally occurring vesicles, microvesicles, ectosomes, nuclei, bacteria, virus, phage, beads, particles, microparticles, nanoparticles, macromolecules, synthetic or naturally occurring lipids or membranes, phospholipid membranes, membrane spheres, the like, or combinations thereof.
  • a sample comprises one or more cells.
  • a sample comprises one or more nuclei.
  • One or more targets may be present in, or on the surface of, or covalently or non-covalently attached to, an organelle, mitochondria, exosomes, liposome, a synthetic or naturally occurring vesicle, a microvesicle, a macrovesicle, an ectosome, a nuclei, a bacteria, a virus, a phage, a bead, a particle, a microparticle, a nanoparticle, a macromolecule, a synthetic or naturally occurring lipid membrane, a phospholipid membrane, or a membrane or lipid sphere.
  • the “ligand” used in these compositions and methods refers to any naturally occurring or synthetic biological or chemical molecule which is used to bind specifically to a single identified target.
  • the binding can be covalently or non-covalent, i.e., conjugated or by any known means taking into account the nature of the ligand and its respective target.
  • first ligand and additional ligand refer to ligands that bind to different targets or different portions of a target. For example, multiple “first ligands” bind to the same target at the same site. Multiple additional ligands bind to a target different than the first ligand and different than any additional ligand.
  • a ligand may independently be selected from a peptide, a protein, an antibody or antibody fragment (e.g., an antigen binding portion of an antibody), an antibody mimetic, an affibody, a ribo- or deoxyribo-nucleic acid sequence, an aptamer, a lipid, a polysaccharide, a lectin, or a chimeric molecule formed of multiples of the same or different ligands.
  • a ligand e.g., a first ligand, and additional ligands, e.g., a second, third, fourth and fifth ligands, etc.
  • a ligand may independently be selected from a peptide, a protein, an antibody or antibody fragment (e.g., an antigen binding portion of an antibody), an antibody mimetic, an affibody, a ribo- or deoxyribo-nucleic acid sequence, an aptamer, a lipid, a polys
  • a ligand include a Fab, Fab′, F(ab′)2, Fv fragment, single-chain Fv (scFv), diabody (Dab), synbody, nanobodies, BiTEs, SMIPs, DARPins, DNLs, Duocalins, adnectins, fynomers, Kunitz Domains Albu-dabs, DARTs, DVD-IG, Covx-bodies, peptibodies, scFv-Igs, SVD-Igs, dAb-Igs, Knob-in-Holes, triomAbs, the like or combinations thereof.
  • a ligand is a recombinant or naturally occurring protein.
  • a ligand is a monoclonal or polyclonal antibody, or fragment thereof.
  • the ligand(s) of the constructs can also be directly labeled with one or more detectable labels, such as fluorophores (see labels discussed below) that can be measured by methods independent of the methods of measuring or detecting the polymer construct described otherwise herein.
  • An antibody fragment or antigen binding fragment of an antibody refers to a portion of an antibody that binds specifically to a target and may include a Fab, Fab′, F(ab′)2, Fv fragment, single-chain Fv (scFv), scFv-Igs, and other fragments or portions of an antibody that can bind specifically to a target.
  • the term “detectable label” means a reagent, moiety or compound capable of providing a detectable signal, depending upon the assay format employed.
  • a label may be associated with the construct as a whole, or with the ligand only, or with the polymer construct or a functional portion thereof. Alternatively, different labels may be used for each component of the construct. Such labels are capable, alone or in concert with other compositions or compounds, of providing a detectable signal.
  • the labels are desirably interactive to produce a detectable signal. Most desirably, the label is detectable visually, e.g. colorimetrically.
  • a variety of enzyme systems operate to reveal a colorimetric signal in an assay, e.g., glucose oxidase (which uses glucose as a substrate) releases peroxide as a product that in the presence of peroxidase and a hydrogen donor such as tetramethyl benzidine (TMB) produces an oxidized TMB that is seen as a blue color.
  • a hydrogen donor such as tetramethyl benzidine (TMB) produces an oxidized TMB that is seen as a blue color.
  • Other examples include horseradish peroxidase (HRP) or alkaline phosphatase (AP), and hexokinase in conjunction with glucose-6-phosphate dehydrogenase that reacts with ATP, glucose, and NAD+ to yield, among other products, NADH that is detected as increased absorbance at 340 nm wavelength.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • hexokinase in conjunction
  • Still other label systems that may be utilized in the described methods and constructs are detectable by other means, e.g., colored latex microparticles (Bangs Laboratories, Indiana) in which a dye is embedded may be used in place of enzymes to provide a visual signal indicative of the presence of the labeled ligand or construct in applicable assays.
  • Still other labels include fluorescent compounds, fluorophores, radioactive compounds or elements.
  • a fluorescent detectable fluorochrome e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), coriphosphine-O (CPO) or tandem dyes, PE-cyanin-5 or -7 (PC5 or PC7)), PE-Texas Red (ECD), PE-cyanin-5.5, rhodamine, PerCP, and Alexa dyes.
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • API allophycocyanin
  • CPO coriphosphine-O
  • tandem dyes PE-cyanin-5 or -7 (PC5 or PC7)
  • PE-Texas Red (ECD) PE-cyanin-5.5
  • rhodamine PE-cyanin-5.5
  • rhodamine PerCP
  • Alexa dyes Alexa dyes.
  • Combinations of such labels such as Texas Red and rhodamine, FITC
  • compositions and methods described herein can also be detectably labeled.
  • the polymer construct(s) can be labeled with one or more detectable labels, such as fluorophores and other labels defined below. The detection of these labels is performed by methods independent of the methods described herein for measurement of the polymer construct or its components.
  • the ligand and polymer construct(s) can be labeled so that when assembled into the final construct, the successful assembly is detectable, such as for production of the final construct.
  • the capture polymer can be labeled with one or more detectable labels.
  • detectable labels can be used in the methods described below, to provide indications of successful binding.
  • the substrate to which the capture polymer is immobilized can be labeled with one or more detectable labels.
  • one or more detectable labels can be used to show successful binding of the capture polymer and the polymer construct.
  • the successful binding of the capture polymer to the substrate can be labeled.
  • the successful association of the polymer construct and the substrate to which the capture polymer is immobilized can be labeled with one or more detectable labels.
  • such labels can be used to indicate the successful association of the ligand and the capture polymer.
  • such labels can be used to indicate the association of the ligand and the substrate to which the capture polymer is immobilized. Still other uses of the detectable labels in these methods and compositions are contemplated.
  • an “antibody or fragment” is a monoclonal antibody, a synthetic antibody, a recombinant antibody, a chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, a multispecific binding construct that can bind two or more targets, a dual specific antibody, a bi-specific antibody or a multi-specific antibody, or an affinity matured antibody, a single antibody chain or an scFv fragment, a diabody, a single chain comprising complementary scFvs (tandem scFvs) or bispecific tandem scFvs, an Fv construct, a disulfide-linked Fv, a Fab construct, a Fab′ construct, a F(ab′)2 construct, an Fc construct, a monovalent or bivalent construct from which domains non-essential to monoclonal antibody function have been removed, a single-chain molecule containing one V L , one V H antigen-binding domain,
  • the “linker” refers to any moiety used to attach or associate the ligand to the polymer construct/oligonucleotide sequence portion of the constructs.
  • the linker is a covalent bond.
  • the linker is a non-covalent bond.
  • the linker is composed of at least one to about 25 atoms.
  • the linker is formed of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 atoms.
  • the linker is at least one to about 60 nucleic acids.
  • the linker is formed of a sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, up to 60 nucleic acids.
  • the linker refers to at least one to about 30 amino acids.
  • the linker is formed of a sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, up to about 30 amino acids.
  • the linker can be a larger compound or two or more compounds that associate covalently or non-covalently. In still other embodiments, the linker can be a combination of the linkers defined herein.
  • the linkers used in the constructs of the compositions and methods are in one embodiment cleavable.
  • the linkers used in the constructs of the compositions and methods are in one embodiment non-cleavable.
  • the linker is a cleavable linker, e.g., disulfide bond or photocleavable bond.
  • the exemplified linker comprises a complex of biotin bound to the construct oligonucleotide sequence by a disulfide bond, with streptavidin fused to the ligand.
  • the biotin is bound to the ligand and the streptavidin is fused to the construct oligonucleotide sequence.
  • the examples shows the exemplified linker bound to the 5′ end of the oligonucleotide of the construct, in other embodiments, the linker may be covalently attached or conjugated other than covalently to any oligonucleotide sequence portion of the construct.
  • the linker when the ligand is a recombinant or synthesized antibody, can be engineered into the antibody sequence to facilitate 1:1 coupling to the polymer construct, thereby simplifying manufacturing of the ligand, the construct and/or the polymer construct.
  • a Halotag® linker can be engineered into the selected ligand (e.g., antibody) or into the polymer construct or component, for such purposes.
  • the ligand is linked to the polymer construct upon production in the same cell. See, e.g., the Halotag® protocols described by Flexi® Vector Systems Technical Manual (TM254 -revised 5/17), copyright 2017 by Promega Corporation; and Janssen D. B., “Evolving haloalkaline dehalogenase”, Curr. Opin. Chem. Biol., 2004, 8:150-159.
  • the “polymer construct” or “construct oligonucleotide sequence” is the portion of the construct which is associated with the ligand. As stated above, this association can be covalent, non-covalent or by any suitable conjugation and employing any suitable linker.
  • the polymer construct is formed by a series of functional polymeric elements, e.g., nucleic acid sequences or other polymers as defined above, each having a function as defined herein.
  • the ligand can be attached to the construct oligonucleotide sequence at its 5′ end or at any other portion, provided that the attachment or conjugation does not prevent the functions of the components of the construct oligonucleotide sequence.
  • the polymer construct can be any length that accommodates the lengths of its functional components.
  • the polymer construct is between 20 and 100 monomeric components, e.g., nucleic acid bases, in length.
  • the construct oligonucleotide sequence is at least 20, 30, 40, 50, 60, 70, 80, 90 or over 100 monomeric components, e.g., nucleic acid bases, in length.
  • the construct oligonucleotide is 200 to about 400 monomeric components, e.g., nucleotides, in length.
  • the polymer construct is generally made up of deoxyribonucleic acids (DNA).
  • the construct oligonucleotide is a DNA sequence.
  • the construct oligonucleotide, or portions thereof comprises modified DNA bases. Modification of DNA bases are known in the art, and can include chemically modified bases including labels.
  • the construct oligonucleotide, or portions thereof comprises ribonucleic acid (RNA) sequences or modified ribonucleotide bases.
  • RNA ribonucleic acid
  • RNA bases can include chemically modified bases including labels.
  • different portions of the construct oligonucleotide sequence can comprise DNA and RNA, modified bases, or modified polymer connections (including but not limited to PNAs and LNAs).
  • the polymer construct is composed of polyamides, PNA, etc.
  • the term “Amplification Handle” refers to a functional component of the construct oligonucleotide sequence which itself is an oligonucleotide or polynucleotide sequence that provides an annealing site for amplification of the construct oligonucleotide sequence.
  • the Amplification Handle can be formed of polymers of DNA, RNA, PNA, modified bases or combinations of these bases, or polyamides, etc.
  • the Amplification Handle is about 10 of such monomeric components, e.g., nucleotide bases, in length.
  • the Amplification Handle is at least about 5 to 100 monomeric components, e.g., nucleotides, in length.
  • the Amplification Handle is formed of a sequence of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96, 97, 98, 99 or up to 100 monomeric components, e.g., nucleic acids.
  • the Amplification Handle when present in first or additional construct oligonucleotide sequences, can be the same or different, depending upon the techniques intended to be used for amplification. In certain embodiments, the Amplification Handle can be a generic sequence suitable as a annealing site for a variety of amplification technologies.
  • Amplification technologies include, but are not limited to, DNA-polymerase based amplification systems, such as polymerase chain reaction (PCR), real-time PCR, loop mediated isothermal amplification (LAMP, MALBAC), strand displacement amplification (SDA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA) and polymerization by any number of DNA polymerases (for example, T4 DNA polymerase, Sulfulobus DNA polymerase, Klenow DNA polymerase, Bst polymerase, Phi29 polymerase) and RNA-polymerase based amplification systems (such as T7-, T3-, and SP6-RNA-polymerase amplification), nucleic acid sequence based amplification (NASBA), self-sustained sequence replication (3SR), rolling circle amplification (RCA), ligase chain reaction (LCR), helicase dependant amplification (HDA), ramification amplification method and RNA-se
  • Barcode or “construct Barcode” describes a defined polymer, e.g., a polynucleotide, which when it is a functional element of the polymer construct, is specific for a single ligand.
  • Barcode can be a “cell Barcode” or “substrate Barcode”, which describes a defined polynucleotide, specific for identifying a particular cell or substrate, e.g., Drop-seq microbead.
  • the Barcode can be formed of a defined sequence of DNA, RNA, modified bases or combinations of these bases, as well as any other polymer defined above.
  • the Barcode is about 2 to 4 monomeric components, e.g., nucleotide bases, in length. In other embodiments, the Barcode is at least about 1 to 100 monomeric components, e.g., nucleotides, in length.
  • the Barcode is formed of a sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96, 97, 98, 99 or up to 100 monomeric components, e.g., nucleic acids.
  • UMI Unique Molecular Identifier
  • RMT Random Molecular Tag
  • the UMI may be inserted into the polymer /construct oligonucleotide sequence as part of the described methods.
  • a UMI is added during the method.
  • another UMI is introduced during reverse transcription.
  • Each UMI is specific for its construct oligonucleotide sequence.
  • each first construct differs only in the sequence of its UMI.
  • Each additional construct will also have its own UMI, which is not present on duplicate additional constructs or additional constructs that differ from each other in target, ligand, Barcode and Anchor specificity.
  • a UMI may be associated with a polymer, e.g., an oligo or polynucleotide sequence, used in a particular assay format or with a polymer, e.g., an oligo or polynucleotide, that is immobilized on a substrate.
  • Each UMI for each polymer construct, e.g., oligonucleotide or polynucleotide is different from any other UMI used in the compositions or methods.
  • the UMI is formed of a random sequence of DNA, RNA, modified bases or combinations of these bases or other monomers of the polymers identified above.
  • a UMI is about 8 monomeric components, e.g., nucleotides, in length.
  • each UMI can be at least about 1 to 100 monomeric components, e.g., nucleotides, in length.
  • the UMI is formed of a random sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96, 97, 98, 99 or up to 100 monomeric components, e.g., nucleic acids.
  • Anchor refers to a defined polymer, e.g., a polynucleotide or oligonucleotide sequence, which is designed to hybridize to another oligonucleotide sequence, e.g., a capture polymer, a capture oligonucleotide, a primer and the like.
  • an Anchor is designed for the purpose of generating a double-stranded construct oligonucleotide sequence.
  • the Anchor is positioned at the 3′ end of an oligonucleotide sequence (e.g., a contruct oligonucleotide sequence).
  • an Anchor is positioned at the 5′ end of a construct oligonucleotide sequence.
  • each Anchor is specific for its intended complementary sequence.
  • an anchor is configured to hybridize to a 3′ end of a capture oligonucleotide such that the 3′ end of the capture oligonucleotide acts as a primer that can generate a second complementary strand of the oligonucleotide in the presence of a polymerase.
  • each first construct has the same Anchor sequence.
  • each additional Anchor has a different additional sequence which hybridizes to a different complementary sequence.
  • each additional Anchor may have the same Anchor sequence as the first or other constructs, depending upon the assay method steps.
  • an Anchor may hybridize to a free complementary sequence or with a complementary sequence that is immobilized on a substrate.
  • the Anchor can be formed of a sequence of monomers of the selected polymer, e.g., DNA, RNA, modified bases or combinations of these bases, PNAs, polyamides, etc.
  • an Anchor is about 3 to 15 monomeric components, e.g., nucleotides, in length.
  • each Anchor can be at least about 3 to 100 monomeric components, e.g., nucleotides, in length.
  • an anchor comprises 3 to 100, 3 to 50, 3 to 30, 5 to 30, 10 to 20, 5 to 20, or 5 to 15 monomeric components (e.g., nucleotides in length).
  • an Anchor is formed of a sequence of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96,
  • Anchor sequence comprises or consists of a polyA sequence.
  • a polyA sequence comprises a nucleic acid sequence comprising ten or more (e.g., 10-40, 10-30 or 10-20) consecutive adenosine nucleotides, derivatives or variants of an adenosine nucleotide, the like, or a combination thereof.
  • an Anchor sequence comprises or consists of a polyT sequence.
  • an Anchor sequence is a polyG sequence.
  • an Anchor sequence may be a random sequence provided that it can hybridize to its intended complementary sequence (e.g., a capture oligonucleotide, amplification primer, or the like).
  • a method described herein may utilize a plurality of oligonucleotides (e.g., a plurality of constructs comprising a ligand attached to an oligonucleotide), where some or all of the oligonucleotides comprise a different anchor (i.e., an anchor having a different nucleic acid sequence, or an anchor having a substantially different nucleic acid sequence).
  • a method described herein may utilize a plurality of oligonucleotides (e.g., a plurality of constructs comprising a ligand attached to an oligonucleotide), where some or all of the oligonucleotides comprise the same anchor.
  • a method described herein may utilize a plurality of oligonucleotides (e.g., a plurality of constructs comprising a ligand attached to an oligonucleotide), where some or all of the oligonucleotides comprise an anchor that is substantially identical (e.g., comprising a nucleic acid sequence that is substantially identical).
  • a method described herein may utilize a plurality of oligonucleotides (e.g., a plurality of constructs comprising a ligand attached to an oligonucleotide), where some or all of the oligonucleotides comprise an anchor comprising a polyA sequence.
  • the polyA sequence of a plurality of anchors is substantially identical. As understood by one of skill in the art, polyA sequences that are substantially identical may differ substantially in length.
  • a polyA sequence e.g, a polyA sequence of an anchor
  • a polyT sequence e.g., an oligonucleotide or capture oligonucleotide comprising a polyT sequence.
  • a polyA sequence may comprise one, two, three or four non-polyA nucleotides and still hybridize efficiently to a polyT sequence, thereby providing an annealed polyA-polyT complex comprising one, two, three or more mismatches.
  • a polyA sequence is a nucleic acid sequence comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% adenosine nucleotides, adenosine analogs, adenosine variants or a combination thereof
  • an oligonucleotide comprises a polyT sequence.
  • a capture oligonucleotide comprises a polyT sequence (e.g., a 3′ polyT sequence).
  • a method described herein may utilize a plurality of oligonucleotides (e.g., a plurality of capture oligonucleotides), where some or all of the oligonucleotides comprise a polyT sequence.
  • a polyT sequence of a plurality of oligonucleotides is substantially identical.
  • a plurality of capture oligonucleotides comprise a polyT sequence that is substantially identical.
  • polyT sequences that are substantially identical may differ substantially in length.
  • a polyT sequence comprises 3 to 100, 3 to 50, 3 to 30, 5 to 30, 10 to 20, 5 to 20, or 5 to 15 consecutive nucleotides (e.g., nucleotides in length).
  • a polyT sequence comprises a nucleic acid sequence comprising three or more, ten or more, 3 to 100, 3 to 50, 3 to 30, 5 to 30, 10 to 20, 5 to 20, or 5 to 15 consecutive thymidine nucleotides, derivatives or variants of a thymidine nucleotide, the like, or a combination thereof.
  • a polyT sequence e.g, a polyT sequence of a capture oligonucleotide
  • a polyT sequence may comprise one, two, three or four non-thymidine nucleotides and still hybridize efficiently to a polyA sequence, thereby providing an annealed polyA-polyT complex comprising one, two, three or more mismatches.
  • a polyT sequence is a nucleic acid sequence comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% thymidine nucleotides, thymidine analogs, thymidine variants or a combination thereof.
  • a polyT sequence comprises one or more uracil nucleotides, or derivative thereof.
  • the “capture oligonucleotide” or “capture oligo” or “capture polymer” is a polymeric sequence, e.g., an oligonucleotide, comprising at least a sequence that is complementary to an Anchor.
  • the capture polymer/oligo is not part of the first or additional constructs; rather it is any polymeric sequence or oligonucleotide belonging to a construct-purification kit or an mRNA-sequencing kit.
  • the term “complementary sequence” refers to a sequence to which an Anchor sequence (or other nucleic acid, e.g., a primer or capture oligonucleotide) is intended to hybridize to, often resulting in a hybridized double stranded complex.
  • an Anchor sequence or other nucleic acid, e.g., a primer or capture oligonucleotide
  • a hybridized complex can often be extended in a 3′ direction where a nucleic acid template is present.
  • a sequence complementary to an anchor can hybridize to an anchor sequence thereby providing a primer for amplification and/or to generate a double stranded sequence.
  • the capture polymer/oligonucleotide sequence may contain sequences that can be used as Amplification Handles and optionally one or more Unique Molecular Identifiers and Barcode sequences.
  • the extension of the capture polymer/oligonucleotide with its complementary sequence hybridized to the Anchor sequence copies the Barcode, the UMI and the Amplification Handle from the first or additional constructs onto the capture polymer/oligonucleotide.
  • the capture polymer/oligonucleotide and its complementary sequence can be formed of DNA, RNA, modified bases or combinations of these bases or of any other polymeric component as defined above.
  • the capture sequence can be unhindered or “free” in the biological sample.
  • the capture polymer/oligo contains a complementary sequence that is a primer sequence designed to participate in amplifying the polymer construct/construct oligonucleotide sequence.
  • the capture sequence is immobilized on a substrate.
  • each capture sequence can be at least about 3 to about 100 monomeric units, e.g., nucleotides, in length.
  • the capture or its complementary sequence is formed of a sequence of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 80, 91, 92, 93, 94, 95, 96, 97, 98, 99 or up to 100 monomeric units, e.g., nucleic acids.
  • a capture oligo contains a complementary sequence polyT sequence when the Anchor sequence is a polyA sequence.
  • the capture oligo contains a polyA sequence.
  • the complementary sequence may be a random polymer, e.g., oligonucleotide sequence, provided that it can hybridize to its intended Anchor sequence.
  • a hashtagging method comprises contacting targets of a sample with one or more constructs comprising a unique barcode that identifies the sample. Where two or more constructs are used in a hashtagging method to tag the same sample, sometimes all of the constructs comprise the same barcode.
  • all of the constructs comprise the same amplification handle, or amplification handles that are substantially identical.
  • the targets used to hashtag multiple sample can be the same or different targets.
  • a first sample is tagged with a first construct
  • a second sample is tagged with a second construct
  • a third sample is tagged with a third construct, where each of the constructs are configured to bind specifically to the same target, however each of the first, second and third constructs comprise a distinguishable barcode that is substantially different.
  • the sample can be pooled for further analysis using a method described herein.
  • the hashtagging allows for later detection, tracking and or quantitation of the each of the samples and targets that are derived from the same sample.
  • one first construct as described above is used to label all cells in a sample prior to pooling multiple samples of cells and prior to performing other scRNA seq or CITE-seq methods using other such constructs having different Amplification Handle sequences.
  • the oligonucleotide portions of the cell hashtag constructs are converted to “hashtags” which enable identification and assignment of each cell within a heterogeneous mixture to its respective original population.
  • the cell-hashtag construct thus serves the purposes of identifying all the cells of a particular sample.
  • the ligand in the cell-hashtag construct can be a pool of antibodies to broadly expressed proteins or a single antibody to such a protein, or any other cell-binding ligand.
  • the Amplification Handle sequence of the cell hashtag is different from that of the first or additional construct used in the CITE-seq methods, one may follow individual cells of an identified sample through the CITE-seq methods, which are typically used to identify cells within a sample that differentially express specific cell surface proteins.
  • immobilized is mean that the capture polymer/oligonucleotide sequence is attached to a solid substrate resulting in reduction or loss of mobility via physical adsorption through charge-charge interation or hydrophobic interaction, covalent bonding, Streptavidin-Biotin interation or affinity coupling. See, e.g. refs 28 and 29.
  • substrate is meant a microparticle (bead), a microfluidics microparticle (bead), a slide, a multi-well plate or a chip.
  • the substrates are conventional and can be glass, plastic or of any conventional materials suitable for the particular assay or diagnostic protocols. See, e.g. refs. 1 and 31.
  • the phrase “consisting essentially of” limits the scope of a described composition or method to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the described or claimed method or composition.
  • a method or composition is described as “comprising” certain steps or features, it is also meant to encompass the same method or composition consisting essentially of those steps or features and consisting of those steps or features.
  • compositions used in the methods described herein comprise one or more of the constructs, first constructs and additional constructs, and a variety of selection of construct components as described above.
  • the selection of the components of the composition will depend upon the identity of the target sought, the RNA sequencing and amplification protocols employed and the purpose of the assay method. In the methods section below, the exemplified methods employ Drop-seq methodologies; however, other methods may be used. The method used may dictate the selection and compositions of the various components described above which make up the composition. Thus the following description of compositions is not exhaustive, and one of skill in the art can design many different compositions based on the teachings provided herein.
  • the composition may also contain the constructs in a suitable carrier or excipient. The elements of each composition will depend upon the assay format in which it will be employed.
  • a composition comprises a “first” construct that comprises a “first” ligand attached or conjugated to a polymer construct, e.g., a construct oligonucleotide sequence, by a linker.
  • the construct oligonucleotide sequence comprises a) an Amplification Handle; b) a Barcode that specifically identifies the first ligand; c) an optional Unique Molecular Identifier that is positioned adjacent to the Barcode on its 5′ or 3′ end; and d) an Anchor (e.g., of at least 3 nucleotides) for hybridizing to a capture oligonucleotide sequence that comprises a sequence complementary to the Anchor.
  • the first ligand binds specifically to a first target located in or on the surface of a cell, such as a cell surface antigen or epitope.
  • a composition comprises multiple substantially identical “first” constructs, wherein each substantially identical first construct differs from the reference “first” construct only in the sequence of the optional Unique Molecular Identifier or its absence from the construct.
  • the composition includes at least one additional construct, which comprises an additional ligand attached or conjugated to an additional construct oligonucleotide sequence by a linker, the additional ligand binding specifically to an additional target located in or on the surface of a cell, and the additional construct oligonucleotide sequence comprising: a) an Amplification Handle; b) an additional Barcode that specifically identifies the additional ligand; c) an optional additional Unique Molecular Identifier that is positioned adjacent to the additional Barcode on its 5′ or 3′ end, and d) an Anchor of at least 3 nucleotides for hybridizing to a complementary sequence.
  • the Amplification Handle or Anchor also differ from the corresponding components in any other construct in the composition.
  • the components specifically identified as “additional” components differ from the corresponding components in any other construct in the composition.
  • a composition comprises multiple substantially identical “additional” constructs, wherein each substantially identical additional construct differs from the reference “additional” construct only in the sequence of the optional Unique Molecular Identifier or its absence from the construct. The number of constructs in a single composition is limited only by the number of targets desired to be identified and/or quantified.
  • the first or additional ligand is an antibody or antibody fragment and the first or additional target is a cell surface epitope.
  • the first or additional ligand is an antibody or antibody fragment and the first or additional target is an intracellular protein.
  • a cell hashtag construct preferably uses a ligand that targets a broadly expressed cellular protein, based on the differences in intended use of these constructs in contrast to the CITE-seq constructs, as described herein.
  • the first construct comprises a first antibody or fragment thereof attached or conjugated to a construct oligonucleotide sequence by a linker, the first antibody or fragment thereof binding specifically to a first epitope sequence located on the surface of a cell, and the construct oligonucleotide sequence comprising: an
  • compositions are particularly suitable wherein the complementary polyT sequence is immobilized on a substrate, e.g., a microfluidics bead.
  • this composition's construct contains a linker that comprises biotin, which is bound to the 5′ end of the construct oligonucleotide sequence by a disulfide bond; and streptavidin, which is fused to the antibody or antibody fragment.
  • Another composition can be designed containing multiple of these first constructs, which differ only in the sequence of the optional Unique Molecular Identifier or its absence from the construct.
  • the composition contains at least one additional construct, which comprises at least one additional antibody or fragment thereof that binds specifically to an additional epitope located in or on the surface of a cell.
  • the additional antibody or fragment is conjugated with an additional construct oligonucleotide sequence by a linker, wherein the additional construct oligonucleotide sequence comprises from 5′ to 3′: an Amplification Handle; an additional Barcode Sequence that specifically identifies the additional antibody or fragment from any other antibody or fragment that recognizes an additional epitope, an optional additional Unique Molecular Identifier sequence that is positioned adjacent to the Barcode on its 5′ or 3′ end, and a polyA sequence of at least 3 nucleotides designed for hybridizing to a polyT sequence, wherein the additional components differ from the corresponding components in any other construct.
  • the Amplification Handle or Anchor differ from the corresponding components in any other construct in the composition.
  • Another exemplary specific composition contains an antibody mimetic as the first ligand and the first target is an intracellularly expressed protein that is present in a biological sample of biopsy tissue.
  • the first construct comprises the antibody mimetic designed for binding to the target protein covalently attached to a construct oligonucleotide sequence by a linker, e.g., a disulfide linker.
  • the construct oligonucleotide sequence comprises in 5′ to 3′ order: an Amplification Handle; a Barcode that specifically identifies the first antibody mimetic; a UMI is positioned adjacent to the Barcode on its 5′ end; and a polyA Anchor sequence.
  • the composition also contains one or more substantially identical first constructs, where each substantially identical first construct differs from the reference “first construct” by containing a different sequence for the UMI. In one embodiment, a substantially identical construct contains no UMI.
  • the composition contains two additional constructs.
  • Each additional construct comprises a different antibody mimetic which specifically binds a different protein present in the biopsied tissue sample.
  • Each of the two additional constructs comprises the antibody mimetic conjugated with its additional construct oligonucleotide sequence by a linker.
  • Each linker can be an optional chemistry as taught above.
  • the construct oligonucleotide sequence comprises from 3′ to 5′: an Amplification Handle; a Barcode Sequence that specifically identifies the additional antibody mimetic from any other antibody or fragment that recognizes a different protein target from the first constructs, and an additional different UMI sequence that is positioned adjacent to the Barcode on its 3′ end, and a polyA sequence of at least 5 nucleotides designed for hybridizing to a polyT sequence.
  • the second additional construct comprises from 5′ to 3′: an Amplification Handle; a Barcode Sequence that specifically identifies an antibody mimetic different from those of the first constructs and from the first additional construct, and which recognizes a third protein target different from the first construct or first additional construct.
  • This second additional construct contains no UMI but contains a polyA sequence of at least 3 nucleotides designed for hybridizing to a polyT sequence.
  • These two additional constructs have targets, antibody mimetic ligands, Barcodes, and UMIs (if present) that differ from each other's corresponding components and differ from the corresponding components in the “first” construct and any substantially identical “first” constructs present in the composition. It should further be understood that compositions may also have one or more substantially identical additional constructs, which differ from the reference additional construct by the UMI, as described above.
  • Kits containing the compositions are also provided. Such kits will contain one or more first or additional constructs, one or more preservatives, stabilizers, or buffers, and such suitable assay and amplification reagents depending upon the amplification and analysis methods and protocols with which the composition will be used. Still other components in a kit include optional reagents for cleavage of the linker, a wash buffer, a blocking solution, a lysis buffer, and an encapsulation solution, detectable labels, immobilization substrates, optional substrates for enzymatic labels, as well as other laboratory items.
  • compositions and kits described above can be used in diverse environments for detection of different targets, by employing any number of assays and methods for detection or targets in general.
  • a method for detecting one or more targets in a biological sample uses the compositions described herein.
  • the method includes the steps of contacting the biological sample with one or more of the compositions described above.
  • the sample is contacted with a composition comprising a first construct that has a first ligand attached or conjugated to a polymer construct, e.g., a construct oligonucleotide sequence, by a linker.
  • the first ligand binds specifically to a first target located in a cell or on the surface of a cell, such as a cell surface epitope.
  • the construct oligonucleotide sequence comprises: an Amplification Handle; a Barcode that specifically identifies the first ligand; an optional Unique Molecular Identifier that is positioned adjacent to the Barcode on its 5′ or 3′ end; and an Anchor for hybridizing to a complementary sequence for generation of a double stranded oligonucleotide sequence.
  • a biological sample is contacted with a composition comprising substantially identical “first” constructs, wherein each substantially identical first construct differs from the reference “first” construct only in the sequence of the optional UMI or its absence from the construct. Therefore the biological sample is contacted with multiple ligands to the same cell surface epitope target.
  • the sample is contacted with a first construct as described above (or multiples thereof); and a composition comprising at least one additional construct.
  • the additional ligand is covalently attached or conjugated to an additional construct oligonucleotide sequence by a linker, the additional ligand binding specifically to an additional target located in a cell or on the surface of a cell.
  • the additional target is in one embodiment a different cell surface epitope.
  • the additional construct oligonucleotide sequence comprising: an Amplification Handle; an additional Barcode that specifically identifies the additional ligand; an optional additional Unique Molecular Identifier that is positioned adjacent to the additional Barcode on its 5′ or 3′ end, and an Anchor of at least 3 nucleotides for hybridizing to a complementary sequence for generation of a double stranded oligonucleotide sequence, wherein the additional components differ from the corresponding components in any other construct in the composition.
  • the Amplification Handle or Anchor differ from the corresponding components in any other construct in the composition.
  • composition may contain one or more substantially identical “additional” constructs, wherein each substantially identical additional construct differs from the reference “additional” construct only in the sequence of the optional UMI or its absence from the construct.
  • the biological sample is washed to remove unbound constructs, if any.
  • the Anchor sequence is then hybridized to its corresponding capture oligo complementary sequence. This can occur by addition of primers as capture complementary sequences or a capture oligo complementary sequence immobilized on a substrate, such as a bead, a slide, a multi-well plate or a chip.
  • the 5′ end of the complementary sequence further comprises: an additional Amplification Handle; an additional Barcode that specifically identifies the substrate to which the capture oligo sequence is bound; and an optional additional Unique Molecular Identifier that is positioned adjacent to the additional Barcode on its 5′ or 3′ end that identifies each capture oligo sequence.
  • the method also includes optionally inserting one or more UMIs to a position adjacent to the Barcode on its 5′ or 3′ end or at any other portion, provided that the insertion does not prevent the functions of the components of the construct oligonucleotide sequence before or after Anchor hybridization.
  • the detection method includes detecting the construct Barcode sequences from each first and additional construct to identify whether the biological sample expresses or contains the first target (e.g., epitope) the additional targets (e.g., one or multiple additional cell surface epitopes) or a combination of the first target and additional targets (e.g., multiple different epitopes).
  • the first target e.g., epitope
  • the additional targets e.g., one or multiple additional cell surface epitopes
  • a combination of the first target and additional targets e.g., multiple different epitopes
  • the expression level of the first target or additional targets in the biological sample is determined by detecting the amount of the corresponding construct Barcodes. In one embodiment, the detection is performed by normalization to the amount of any one of Unique Molecular Identifiers or the mean amount of two or more of Unique Molecular Identifiers.
  • Various embodiments of the methods can include adding to the biological sample the composition containing the first construct(s) only, or compositions containing additional construct(s) simultaneously or sequentially prior to the washing step. Further method steps can include isolating the biological sample into individual cells or populations of cells before the contacting step or after the washing step. Another step involves extending the capture oligonucleotide hybridized to the Anchor sequence to copy a construct barcode, UMI and Amplification handle onto double stranded sequences. The double-stranded oligonucleotide sequences can also be generated after anchor hybridization with primers annealed to the Amplification handles after either anchor hybridization and/or insertion of UMIs.
  • an oligonucleotide comprises one or more barcodes.
  • Any suitable barcode can be used for a composition or method described herein.
  • a barcode often comprises or consists of a relatively short nucleic acid sequence of, for example, 2 to 50, 2 to 30, 2 to 20, 2 to 15, 10-20, 4 to 15, or 2 to 5 consecutive nucleotides, where the nucleotide sequence of a barcode is unique to a nucleic acid, an oligonucleotide, a population of oligonucleotides (e.g., a plurality of substantially identical oligonucleotides), a population of substantially identical constructs, a sample, a sample source, a ligand, a lot, a run, or a combination thereof.
  • a barcode that is unique to an oligonucleotide attached to a first ligand can be used to identify the presence of, or amount of the first ligand after detection of the barcode sequence (e.g., by sequencing, or another suitable method).
  • a barcode that is unique to an oligonucleotide attached to a first ligand can be used to specifically identify the presence, amount or absence of a ligand (e.g., a first ligand, a second ligand or an additional ligand) in a multiplex sample comprising other ligands, other nucleic acids, other oligonucleotides and other barcodes.
  • Such a barcode may be termed a “ligand-specific” barcode, or a barcode that specifically identifies a ligand (e.g., a first ligand, or any specific ligand).
  • a barcode that is unique to an oligonucleotide, sample, bead, lot, or run can be used to specifically identify a particular oligonucleotide, sample, bead, lot, or run in a multiplex sample comprising a plurality of other oligonucleotides, samples, beads, lots, or runs.
  • a barcode that can be used to specifically identify a sample may be termed a “sample-specific” barcode, or a barcode that specifically identifies a sample.
  • a barcode that can be used to specifically identify a bead may be termed a “bead-specific” barcode, or a barcode that specifically identifies a bead.
  • a barcode that can be used to specifically identify an oligonucleotide or nucleic acid may be termed a “oligonucleotide-specific” barcode or a “nucleic acid-specific” barcode, respectively.
  • an oligonucleotide comprises a unique molecular identifier (UMI).
  • a UMI comprises a unique barcode that specifically identifies an individual oligonucleotide from all other oligonucleotides used in a composition or method described herein.
  • Methods and compositions described herein can be used, in some embodiments, to determine the presence, amount, or absence of a sample, target, construct or oligonucleotide.
  • determining the amount of a sample, target, construct or oligonucleotide comprises determine an absolute, approximate, mean, average or relative amount of a sample, target, construct or oligonucleotide in a multiplex assay. Accordingly, in certain embodiments, methods and compositions described herein can be used to quantitate amounts of a sample, target, construct or oligonucleotide in a multiplex assay.
  • an oligonucleotide comprises an amplification handle. Any suitable amplification handle can be used for a composition or a method described herein. In some embodiments, an amplification handle comprises a relatively short length of consecutive amino acids that is integrated into an oligonucleotide, or nucleic acid described herein. An amplification handle can be any suitable length. In some embodiments, an amplification handle is 5 to 50, 5 to 40, 5 to 35, 5 to 25 or 5 to 15 nucleotides in length. In certain embodiments, an amplification handle is used for capture and/or amplification and/or sequencing of a nucleic acid.
  • An amplification handle may comprise any nucleic acid sequence suitable for primer binding, capture, extension by a polymerase, and/or amplification by a polymerase.
  • an amplification handle comprises a primer binding site.
  • a primer comprises a nucleic acid sequence substantially identical to an amplification handle.
  • an oligonucleotide comprises an interposed nucleic acid flanked by a 5′ and 3′ amplification handle, or complement thereof, wherein the flanking amplification handles facilitate amplification of the interposed nucleic acid.
  • Another variation of this method involves cleaving the ligand from the construct prior to or after Anchor hybridization to a complementary sequence. Still another embodiment involves lysing the cell, when desired.
  • the lysis technique can involve exposure of the cells to detergents, detergent-buffer solutions, such as RIPA buffer, IP-lysis buffers, M-PER or B-PER reagent solutions (Pierce Chemical) and the like.
  • the ligand-oligonucleotide constructs can be used with targets other than cell surface antibodies and ligands other than antibodies as discussed herein.
  • a further embodiment involves cell permeabilization and an optional fixation procedure before the contacting step or between sequential contacting steps with first or additional constructs.
  • the permeabilizing technique can involve exposure of the biological samples to organic solvents (for example but not limited to methanol andacetone), detergents (such as SaponinTM, Triton X-100TM and Tween-20TM), other reagent available to one of skill in the art(such as Zinc Salt Solution 32 , eBioscienceTM Intracellular Fixation & Permeabilization Buffer Set and FIX & PERM® Cell Fixation & Cell Permeabilization Kit) and any combination thereof.
  • the fixation step is optional before or during the permeabilization.
  • fixation for example contacting the biological samples with solution containing crosslinking fixatives (such as formaldehyde, glutaraldehyde and other aldehyde), precipitating fixatives (such as methanol, ethanol, acetone and acetic acid), oxidizing agents (such as osmium tetroxide, potassium dichromate, chromic acid and potassium permanganate), mercurials, picrates, Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE) fixative, 2,4,6-Trimethylpyridine, eBioscienceTM Intracellular Fixation & Permeabilization Buffer Set, FIX & PERM® Cell Fixation & Cell Permeabilization Kit or any combination thereof.
  • crosslinking fixatives such as formaldehyde, glutaraldehyde and other aldehyde
  • precipitating fixatives such as methanol, ethanol, acetone and acetic acid
  • oxidizing agents such as osmium
  • detection protocols including without limitation, PCR, Immuno-PCR 15 and proximity ligation or proximity extension assay 16 protocols, PEA 26 , RCA 25 , sequencing and fluorescence hybridization protocols.
  • the method is a high-throughput method.
  • the compositions described herein are used in high-throughput protocols such as the following.
  • a high-throughput method for detecting one or more epitopes in a biological sample can employ hundreds or thousands of wells containing the same or different samples.
  • the method comprising contacting a biological sample with a composition comprising a first construct that comprises a first antibody or fragment thereof that binds specifically to a first epitope, the first antibody or fragment attached or conjugated to an construct oligonucleotide sequence by a linker, wherein the construct oligonucleotide sequence comprises: an Amplification Handle, a Barcode Sequence that specifically identifies the first antibody or fragment from any other antibody or fragment that recognizes a different epitope, an optional Unique Molecular Identifier sequence that is positioned adjacent to the Barcode on its 5′ or 3′ end, and an Anchor sequence (e.g., of at least 3 nucleotides) for hybridizing to a complementary sequence for generating a double-stranded oligonucleotide sequence.
  • a composition comprising a first construct that comprises a first antibody or fragment thereof that binds specifically to a first epitope, the first antibody or fragment attached or conjugated to an construct oligonucleotide sequence by a
  • the composition comprises one or more substantially identical constructs, wherein each substantially identical first construct differs only in the sequence of the optional Unique Molecular Identifier or its absence from a reference (e.g., “first” or “additional”) construct.
  • the composition comprises at least one additional construct, which comprises an additional antibody or fragment thereof attached or conjugated to an additional construct oligonucleotide sequence by a linker. The additional antibody or fragment thereof binds specifically to an additional epitope.
  • the additional construct oligonucleotide sequence comprises: the same or different Amplification Handle, an additional Barcode that specifically identifies the additional antibody or fragment thereof; an optional additional Unique Molecular
  • Identifier that is positioned adjacent to the additional Barcode on its 5′ or 3′ end, and the same or different Anchor, wherein the additional target and the additional antibody or fragment ligand, optional UMI, and additional Barcode components differ from the corresponding components in any other construct in the composition.
  • High-throughput protocols also involve washing the biological sample to remove unbound constructs; annealing the construct oligonucleotide sequence(s) through their respective Anchors to the corresponding complementary sequences and generating double stranded oligonucleotide sequence(s).
  • UMIs may also be optionally inserted to a position adjacent to the Barcode on its 5′ or 3′ end before or after Anchor hybridization.
  • Such methods involve detecting the construct Barcode sequence(s) to identify whether the biological sample (or samples present in individual wells) expresses or contains the first target, the additional targets, or a combination of first target and additional targets.
  • expression level of the first target or additional targets in the biological sample occurs by detecting the amount of the corresponding Barcodes.
  • the said detection is performed by normalizing to the amount of a Unique Molecular Identifier or the mean amount of two or more Unique Molecular Identifiers.
  • the high-throughput methods also can include addingone or more first and additional constructs to a biological sample simultaneously or sequentially prior to the washing step.
  • the methods can also include isolating the biological sample(s) bound to one or more of the first or additional constructs into individual cells or populations of cells after washing; and amplifying the double strand oligonucleotide sequence with primers annealed to Amplification Handles. Any of the other parameters of the compositions can be included that coordinate with the assay protocols used in the detection.
  • compositions described herein are designed and used to overcome the limitations of the currently existing methods for detecting and/or measuring RNA transcripts and proteins in single cells (i.e., droplet technology).
  • the method referred to as Cellular Indexing of Transcriptome and Epitopes by sequencing (CITE-seq) uses the compositions described herein to simultaneously characterize the transcriptome and a potentially unlimited number of cell-surface markers from the same cell in a high-throughput manner. It combines unbiased genome-wide expression profiling with the measurement of specific protein markers in thousands of single cells using droplet microfluidics.
  • the compositions can be used, in addition to adding an extra dimension to single-cell transcriptome data. This method provides a more detailed characterization of cell populations, but also allows study of post-transcriptional (and post-translational) gene regulation in single cells at an unprecedented depth.
  • a suspension of mixed human and mouse cells and the Drop-seq protocol were employed with constructs comprising monoclonal antibodies as construct ligands attached to construct oligonucleotides containing the unique antibody identifier sequences (Barcodes).
  • the cell suspension is labeled with the ligand—oligonucleotide sequence constructs (in these case oligo-tagged antibodies) and single cells are subsequently encapsulated into nanoliter-sized aqueous droplets in a microfluidic apparatus.
  • antibody and cDNA molecules are indexed with the same unique Barcode and are converted into libraries that are amplified independently and mixed in appropriate proportions for sequencing in the same lane.
  • the inventors were able to unambiguously identify human and mouse cells based on their species-specific cell surface proteins and independently on their transcriptome.
  • CITE-seq allows in-depth characterization of single cells by simultaneous measurement of gene-expression levels and cell-surface proteins, is highly scalable, only limited by the number of specific antibodies that are available and is compatible with other single-cell sequencing systems.
  • a single cell sequencing platform suitable for integration with the compositions and methods described herein is the Drop-seq method, including, but not limited to, microfluidic, plate-based, or microwell, Seq-WellTM method 35 and adaptations of the basic protocol, and InDropTM method 2 (1 Cell Bio).
  • a single cell sequencing platform suitable for integration with the compositions and methods described herein is 10 ⁇ genomics single cell 3′ solution (www.10 ⁇ genomics.com/single-cell/) 3 , or single cell V(D)J solution (www.10xgenomics.com/vdj/, either run on Chromium controller, or dedicated Chromium single cell controller).
  • Still other useful sequencing protocols for combination with CITE-seq as described herein include Wafergen iCell8TM method 3, 38-40 (www.wafergen.com /products/ice118-single-cell-system); Microwell-seq method 41 , Fluidigm C1TM method 42-44 and equivalent single cell products.
  • Still other known sequencing protocols useful with the compositions and methods described herein include BD ResolveTM single cell analysis platform 37 (derived from Cyto-seq) and ddSeq 6 (from Illumina® Bio-Rad® SureCellTM WTA 3′ Library Prep Kit for the ddSEQTM System, 2017, Pub. No.
  • compositions and methods described herein are useful with combinatorial indexing based approaches (sci-RNA-segTM method 20 or SPLiT-seqTM method 30 ) and Spatial Transcriptomics, or comparable spatially resolved sequencing approaches 36 .
  • the methods and compositions described herein can also be used as an added layer of information on standard index sorting (FACS) and mRNA-sequencing-based approaches.
  • FACS standard index sorting
  • mRNA-sequencing-based approaches In one embodiment, for example, standard FACS panels are supplemented with other CITE-seq tagged antibodies detectable through plate-based sequencing. Still other sequencing protocols can be combined with the compositions and methods specifically described herein.
  • nucleic acid sequencing method can be used to sequence the nucleic acids described herein and/or to detect the presence, absence or amount of the various nucleic acids, constructs, targets, oligonucleotides, amplification products and barcodes described herein.
  • a high-throughput method for characterizing a cell by simultaneous detection of one or more epitopes located in or on the cell and the transcriptome involves contacting a biological sample containing cells with one or more of the composition as above described.
  • a composition that comprises a first antibody or fragment thereof that binds specifically to a first epitope located in or on the surface of a cell, the first antibody or fragment is conjugated to a construct oligonucleotide sequence by a linker, wherein the construct oligonucleotide sequence comprises: an Amplification Handle; a Barcode Sequence that specifically identifies the first antibody or fragment from any other antibody or fragment that recognizes a different epitope, an optional Unique Molecular Identifier sequence that is positioned adjacent to the Barcode on its 5′ or 3′ end, and a polyA sequence of at least 3 nucleotides designed for hybridizing to a polyT sequence immobilized on a microfluidics bead.
  • the composition comprises one or more substantially identical
  • the composition further comprises at least one additional construct, which comprises an additional antibody or fragment thereof conjugated to an additional construct oligonucleotide sequence by a linker, the additional antibody or fragment thereof binding specifically to an additional epitope, and the additional construct oligonucleotide sequence comprising from 5′ to 3′: the Amplification Handle; an additional Barcode that specifically identifies the additional antibody or fragment thereof; an optional additional Unique Molecular Identifier that is positioned adjacent to the additional Barcode on its 5′ or 3′ end, and the Anchor, wherein the additional components differ from the corresponding components in any other construct in the composition.
  • the compositions can be added to the biological sample simultaneously or sequentially prior to a washing step.
  • the composition comprises one or more substantially identical “additional” constructs, wherein each substantially identical additional construct differs only in the sequence of the optional Unique Molecular Identifier or its absence from the reference “additional” construct.
  • an individual single cell bound to one or more constructs is encapsulated into an aqueous droplet with one bead, wherein each bead is conjugated to a construct comprising a unique cell Barcode sequence comprising a 3′ polyT sequence.
  • the single cell in each droplet is lysed, wherein mRNAs in the cell and the construct oligonucleotide from the antibody or fragment anneal to the polyT sequences of the bead.
  • This method can also include a step of optionally inserting one or more Unique Molecular Identifiers to a position adjacent to the additional Barcode on its 5′ or 3′ end before or after the annealing or hybridization step.
  • such a method involves creating by amplification a library containing the cDNA from the target cell's transcriptome, and the DNA containing the construct oligonucleotide sequence(s).
  • the construct Barcode sequences are detected to identify whether the single cell expresses the first epitope.
  • the expression level of the first epitope in the single cell is determined by detecting the amount of the construct Barcode.
  • the detection is performed by normalization of the amount of any of the Unique Molecular Identifiers or the mean amount of two or more Unique Molecular Identifiers.
  • the transcriptome of the library is associated with the cell identified by the binding and identification of the first and/or additional constructs.
  • compositions and methods described herein provide in one aspect a sequencing-based method that combines highly multiplexed ligand-based (e.g., antibody-based) detection of well-established protein markers together with unbiased transcriptome profiling for thousands of single cells in parallel.
  • highly multiplexed ligand-based e.g., antibody-based
  • the examples demonstrate a novel method that can profile many targets, e.g., cellular markers and single-cell transcriptomes on thousands of cells in parallel.
  • the further analysis includes analysis by any of the methods described herein.
  • the oligonucleotide portions of the cell hashtag constructs, particularly the Amplification Handle sequences, are different from those used in the “further” analytic methods, which permit cell hashtagging of samples subjected to those methods.
  • This “hashtagging” method performed prior to pooling of samples subjected to additional analyses has several advantages. Multiplexing enables cost savings and the ability to control for batch effects—for example, process treated/untreated at the same time.
  • the cell hashtag constructs allow unequivocal determination of most doublets. Finally, the combination of these two advantages allows us to vastly overload droplet- based scRNA-seqexperiments (i.e., use 20,000 cells, rather than 4,000 cells, per lane), resulting in decreased cost of experiments and increased information produced by the experiments.
  • This hashtagging embodiment can be used to multiplex samples of the same genotype without the need to perform genotyping on samples.
  • the hashtagging methods can be extended to barcoding or identifying nuclei as well as other cellular components.
  • Still further embodiments follow as “A1 through “E36”.
  • a capture sequence comprising a polyA sequence of at least 3 nucleotides designed for hybridizing to a polyT sequence
  • composition comprising a first construct that comprises a first ligand attached or conjugated to a polymer construct by a linker, said first ligand binding specifically to a first target, and said polymer construct comprising: an Amplification Handle; a Barcode that specifically identifies said first ligand; an optional Unique Molecular Identifier that is positioned adjacent to the Barcode on its 5′ or 3′ end; and an Anchor for hybridizing to a capture sequence that comprises a sequence complementary to said Anchor;
  • composition comprising at least one additional construct, which construct comprises an additional ligand attached or conjugated to an additional polymer construct by a linker, said additional ligand binding specifically to an additional target, and said additional polymer construct comprising an Amplification Handle; an additional Barcode that specifically identifies said additional ligand; an optional additional Unique Molecular Identifier that is positioned adjacent to the additional Barcode on its 5′ or 3′ end, and an Anchor for hybridizing to a capture sequence that comprises a sequence complementary to said Anchor; and
  • composition comprising one or more substantially identical constructs, each substantially identical construct differing from any other reference first or additional construct in the sequence of its optional Unique Molecular Identifier (UMI) or the absence of the UMI.
  • UMI Unique Molecular Identifier
  • composition comprising a first construct that comprises a first antibody or fragment thereof that binds specifically to a first epitope, said first antibody or fragment attached or conjugated to a first polymer construct by a linker, wherein the first polymer construct comprises: an Amplification Handle; a Barcode Sequence that specifically identifies said first antibody or fragment from any other antibody or fragment that recognizes a different epitope, an optional Unique Molecular Identifier sequence that is positioned adjacent to the 5′ or 3′ end of the Barcode, and an Anchor sequence for hybridizing to a capture sequence that comprises a sequence complementary to said Anchor;
  • composition of (i) comprising at least one additional construct, which comprises an additional antibody or fragment thereof attached or conjugated to an additional polymer construct by a linker, said additional antibody or fragment thereof binding specifically to an additional epitope, and said additional polymer construct comprising: an Amplification Handle; an additional Barcode that specifically identifies said additional antibody or fragment thereof; an optional additional Unique Molecular Identifier that is positioned adjacent to the 5′ or 3′ end of the additional Barcode, and an Anchor sequence of (i), wherein said additional construct differs from any other construct in the composition in its antibody, epitope, Barcode, and UMI; and
  • iii a composition of (i) or (ii) comprising one or more substantially identical constructs, each substantially identical construct differing from any other reference first or additional construct in the sequence of its optional Unique Molecular Identifier (UMI) or the absence of the UMI.
  • UMI Unique Molecular Identifier
  • composition that comprises a first construct that comprises a first antibody or fragment thereof that binds specifically to a first epitope located in or on the surface of a cell, said first antibody or fragment conjugated to a first polymer construct by a linker, wherein the first polymer construct comprises an Amplification Handle; a Barcode Sequence that specifically identifies said first antibody or fragment from any other antibody or fragment that recognizes a different epitope, an optional Unique Molecular Identifier sequence that is positioned adjacent the 5′ or 3′ end of the Barcode, and a polyA Anchor sequence designed for hybridizing to a capture oligonucleotide sequence comprising a polyT sequence immobilized on a microfluidics bead;
  • composition of (i) comprising at least one additional construct, which comprises an additional antibody or fragment thereof conjugated to an additional polymer construct by a linker, said additional antibody or fragment thereof binding specifically to an additional epitope, and said additional polymer construct comprising: the Amplification Handle of (i); an additional Barcode that specifically identifies said additional antibody or fragment thereof; an optional additional Unique Molecular Identifier that is positioned adjacent to the 5′ or 3′ end of the additional Barcode, and the said Anchor of (i), wherein said additional antibody or fragment, additional Barcode, additional UMI and additional epitope differ from the corresponding components in any other construct in the composition; and
  • iii a composition of (i) or (ii) comprising one or more substantially identical constructs, each substantially identical construct differing from any other reference first or additional construct in the sequence of its optional Unique Molecular Identifier (UMI) or the absence of the UMI.
  • UMI Unique Molecular Identifier
  • each bead is conjugated to the capture sequence comprising a unique bead Barcode sequence, an optional UMI, and a 3′ polyT sequence.
  • a fourth construct comprising a third ligand attached to a fourth oligonucleotide, wherein the third ligand binds specifically to a third target, and the fourth oligonucleotide comprises:
  • a) a first construct comprising a first ligand attached to a first oligonucleotide, wherein the first ligand binds specifically to a first target, and the first oligonucleotide comprises:
  • a second construct comprising the first ligand attached to a second oligonucleotide, wherein the second oligonucleotide comprises:
  • a third construct comprising a second ligand attached to a third oligonucleotide, wherein the second ligand binds specifically to a second target, and the third oligonucleotide comprises:
  • a fourth construct comprising a third ligand attached to a fourth oligonucleotide, wherein the third ligand binds specifically to a third target, and the fourth oligonucleotide comprises:
  • a unique barcode configured to specifically identify a first sample
  • an anchor comprising a polyA sequence.
  • a fourth construct comprising a third ligand attached to a fourth oligonucleotide, wherein the third ligand binds specifically to a third target, and the fourth oligonucleotide comprises:
  • a) a first construct comprising a first ligand attached to a first oligonucleotide, wherein the first ligand binds specifically to a first target, and the first oligonucleotide comprises:
  • a second construct comprising the first ligand attached to a second oligonucleotide, wherein the second oligonucleotide comprises:
  • a third construct comprising a second ligand attached to a third oligonucleotide, wherein the second ligand binds specifically to a second target, and the third oligonucleotide comprises:
  • a fourth construct comprising a third ligand attached to a fourth oligonucleotide, wherein the third ligand binds specifically to a third target, and the fourth oligonucleotide comprises:
  • a unique barcode configured to specifically identify a first sample
  • an anchor comprising a polyA sequence.
  • Antibody-oligos were designed with the following characteristics: a generic Amplification Handle (PCR handle) for next-generation sequencing library preparation, a unique Barcode sequence specific for each antibody, and a polyA stretch at the 3′ end ( FIG. 1A ). Two antibody-oligos were generated. Anti-Mouse Integrin Beta-1 (CD29) antibodies were linked to Barcoded oligo 1 containing a disulfide bridge, a common sequence (Amplification Handle, PCR handle), a unique antibody identifier Barcode (5′-ATGTCCT-3′) and a UMI containing 4 nt followed by a polyA tail ( FIG. 2B , top panel).
  • PCR handle PCR handle
  • Barcode sequence specific for each antibody a polyA stretch at the 3′ end
  • FIG. 1A Two antibody-oligos were generated.
  • Anti-Mouse Integrin Beta-1 (CD29) antibodies were linked to Barcoded oligo 1 containing a disulfide bridge, a common sequence (Amplification Handle,
  • Anti-human CD29 antibodies were linked to Barcoded oligo 2 containing a disulfide bridge, a common sequence (Amplification Handle, PCR handle), a unique antibody identifier Barcode (5′-GCCATTA-3′) and a UMI containing 4 nt followed by a polyA tail ( FIG. 2B , bottom panel).
  • the oligos were modified with biotin and a disulfide bond at the 5′ end of the oligo and were bound to streptavidin modified antibodies.
  • the oligo could be released from the antibody by reducing the disulfide bond.
  • SAV streptavidin-biotin
  • a commercially available kit to streptavidin label antibodies (generally used for subsequent fluorophore labelling for FACS) was used.
  • Antibodies were linked to biotinylated oligos ( FIG. 2A , Lane and Panel # 1 ).
  • FIG. 2A Lane and Panel # 2
  • Other attachment chemistries are useful, including but not limited to thiol-maleimide, thiol-haloacetate, amine-NHS, amine-isothiocyanate, azide-alkyne (CuAAC), tetrazole-cyclooctene (iEDDA, used in Example 7, 45 (refs. 25, 45 and 46 and references therein), and can be cleavable or non-cleavable covalent linkages.
  • DNA oligonucleotides with a 5′ amine modification were purchased at IDT (USA) and biotinylated using NHS-chemistry according to manufacturer's instructions (EZ Biotin S-S NHS, Thermo Fisher Scientific, USA).
  • EZ Biotin S-S NHS Thermo Fisher Scientific, USA.
  • the optional disulfide bond allows separation of the oligo from the antibody with reducing agents in some embodiments. Separation of the oligo from the antibody may not be needed for all applications.
  • Excess Biotin-NHS was removed by gel filtration (Micro Biospin 6, Bio-Rad) and ethanol precipitation.
  • Streptavidin-labeled antibodies were incubated with biotinylated oligonucleotides in equimolar ratio (assuming two streptavidin tetramers per antibody on average) overnight at 4° C. in PBS containing 0.5 M NaCl and 0.02% Tween. Unbound oligo was removed from antibodies using centrifugal filters with a 50 KDa MW cutoff (Millipore, USA). Removal of excess oligo was verified by 4% agarose gel electrophoresis. Antibody-oligo conjugates were stored in PBS supplemented with sodium azide (0.05%) and BSA (1 ⁇ g/ ⁇ l) at 4° C.
  • RNA-seq protocols including 10 ⁇ Genomics and Drop-seq
  • Single cell RNA-seq protocols also rely entirely on this activity to append a PCR handle to the 5′ end of full-length cDNAs.
  • the PCR handle is used for subsequent amplification.
  • the PCR amplification handle in the antibody-barcoding oligos must be changed depending on which sequence read is used for RNA readout (e.g., 10 ⁇ Single Cell 3′ vl uses read 1, while Drop-seq and 10x Single Cell 3′ v2 use read 2).
  • UMIs which are redundant for Drop-seq and 10 ⁇ protocols due to the UMI addition to the cDNA at reverse transcription.
  • UMIs on the antibody-conjugated oligonucleotide may be useful for other iterations of the method where UMIs are not part of the scRNA-seq library preparation protocol.
  • BC6 SEQ ID NO: 1 /5AmMC12/GTCTCGTGGGCTCGGAGATGTGTATA AGAGACAGGCCAATNNBAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA BC12: SEQ ID NO: 2 /5AmMC12/GTCTCGTGGGCTCGGAGATGTGT ATAAGAGACAGCTTGTANNBAAAAAAAAAAAAAAAAAAAAAAA Species mixing, 10x (single cell 3′ version 1, Nextera read1 handle).
  • BC6 SEQ ID NO: 3 /5AmMC12/TCGTCGGCAGCGTCAGATGT GTATAAGAGACAGGCCAATNNBAAAAAAAAAAAAAAAAAAAAAAAAAA BC12: SEQ ID NO: 4 /5AmMC12/TCGTCGGCAGCGTCAGATGTGTATA AGAGACAGCTTGTANNBAAAAAAAAAAAAAAAAAAAAAAA CBMC profiling-(Drop-seq and 10x v2 compatible oligos, containing TruSeq small RNA read 2 handle).
  • Drop-seq was performed as described with modifications.
  • cells were loaded at a concentration of 400 cells/4 to achieve a high doublet rate.
  • PBMC experiments cells were loaded at 150 cells/4.
  • cDNA was amplified for ten cycles, and products were then size separated with Ampure Beads (Beckman Coulter, USA) into ⁇ 300 nt fragments containing antibody-derived tags (ADTs) and >300 nt fragments containing cDNAs derived from cellular mRNA.
  • ADTs were amplified for ten additional cycles using specific primers that append P5 and P7 sequences for clustering on Illumina flowcells.
  • antibody tags can be amplified directly from thoroughly washed Dropseq beads after RNA-cDNA amplification using specific primers for the antibody oligo and Drop-seq bead-RT oligo. cDNAs derived from mRNA were converted into sequencing libraries by tagmentation as described 1 . After quantification, libraries were merged at desired concentrations (10% of a lane for ADT, 90% cDNA library). Sequencing was performed on a HiSeq 2500 Rapid Run with v2 chemistry per manufacturer's instructions (Illumina, USA).
  • the 10 ⁇ single-cell run was performed according to the manufacturer's instructions (10 ⁇ Genomics, USA) with modifications.
  • human/mouse mixing experiment (run on Single Cell 3′ version 1) ⁇ 17,000 cells were loaded to yield ⁇ 10,000 cells with an intermediate/high doublet rate.
  • CBMC profiling (run on Single Cell 3′ version 2), ⁇ 7,000 cells were loaded to obtain a yield of ⁇ 4,000 cells.
  • CBMC profiling we spiked-in mouse cells at low frequency ( ⁇ 4%). This allowed us to draw antibody signal-to-noise cutoffs and to estimate the true doublet rates (4%) in our experiments and compare these rates to the estimates provided by the equipment manufacturer ( ⁇ 3.1%) (see below).
  • cDNA was amplified for ten cycles, and products were then size separated with Ampure Beads (Beckman Coulter, USA) into ⁇ 300 nt fragments containing antibody-derived tags (ADTs) and >300 nt fragments containing cDNAs derived from cellular mRNA.
  • ADTs were amplified for ten additional cycles using specific primers that append P5 and P7 sequences for clustering on Illumina flowcells.
  • a sequencing library from cDNAs derived from RNA was generated using a tagmentation-based approach akin to that used in Drop-seq for the Single Cell 3′ v1 experiments, or according to manufacturer's instructions for the Single Cell 3′ v2 experiments.
  • ADT and cDNA libraries were merged and sequenced as described above.
  • HeLa (human), 4T1 (mouse) and 3T3 (mouse) cells were maintained according to standard procedures in Dulbecco's Modified Eagle's Medium (Thermo Fisher, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher, USA) at 37 ° C. with 5% CO2.
  • FBS fetal bovine serum
  • HeLa and 4T1 cells were mixed in equal proportions and incubated with DNA barcoded CITE-seq antibodies as described above.
  • ⁇ 5% 3T3 cells were mixed into CBMC pool before performing CITE-seq.
  • CBMCs Cord blood mononuclear cells
  • Cells were stained with a mixture of fluorophore (CD8a-FITC, BioLegend, USA) labeled antibodies and CITE-seq oligo-labeled antibodies from the same monoclonal antibody clone (RPA-T8) targeting CD8a, at concentrations recommended by the manufacturer (1 ug per test, BioLegend, USA). Cells were also stained with Anti-CD4-APC antibody (RPA-T4, BioLegend, USA). Cells were sorted into pools of different CD8a expression levels using the Sony SH800 cell sorter, which was operated per manufacturer's instructions. Pools were then split into two and reanalyzed by flow cytometry using Sony SH800 or processed for CITE-seq using Drop-seq as described above. Flow cytometry data were plotted using FlowJo v9 (USA).
  • CD3e clone SK7 Hilyte 750 Allophycocyanin (H7APC)
  • CD4 clone SK3 Brilliant Blue
  • CD8a clone SK1) Phycoerythrin (PE)
  • CD14 clone M5E2 Brilliant Blue
  • Antibody and cell barcodes were directly extracted from the reads in the fastq files. Since the antibody barcodes were sufficiently different in the species mixing experiment, we also counted sequences with Hamming distance less than 4. For the CBMCs we counted sequences with Hamming distance less than 2. Reads with the same combination of cellular, molecular and antibody barcode were only counted once. We kept only cells that passed the RNA-specific filters and had a minimum number of total ADT counts (minimum counts used: species mixing, 10; CD8a FACS comparison, 1; CBMC, 50).
  • ci,j is the molecule count of gene i in cell j
  • mj is the sum of all molecule counts for cell j.
  • the clustering algorithm as implemented in the “cluster_louvain” function of the igraph R package, find a partitioning of the cells with high density within communities as compared to between communities. For 2D visualization we further reduced the dimensionality of the data to 2 using t-SNE 58,34,59 .
  • Example 1 The antibody-oligo complexes described in Example 1 were incubated with cells using conditions established for flow cytometry, such as ref 22. The cells were washed to remove unbound antibodies, then single cells were encapsulated into nanoliter-sized aqueous droplets in a microfluidic apparatus designed to perform Drop-seq 1 ( FIG. 1C ). After cell lysis (which happened immediately in the droplets when the lysis buffer contacted cells), cellular mRNAs annealed to polyT containing Drop-seq beads ( FIG. 1B ) via their polyA tail ( FIG. 1C # 6 ). Oligos from the antibodies also annealed to the Drop-seq beads via their poly-A stretch at the 3′ end.
  • a unique Barcode sequence on the Drop-seq bead indexed the transcriptome of each co-encapsulated cell. After breaking the emulsion and removing the oil, reverse transcription extended the Barcoded oligo to create the first-strand cDNA from both mRNA and antibody-derived oligo templates.
  • the cDNA and the antibody-derived tags were separated by size ( FIG. 2C ) and converted into Illumina-ready libraries independently. The two library types were sequenced together. Due to the advantages of generating libraries separately, relative proportions of the libraries are also tailored to ensure appropriate sequencing depth is obtained.
  • This method is expanded to simultaneously measure large numbers of established antibody-based cell markers and transcriptome in tens of thousands of cells in parallel.
  • a further experiment yields qualitative cell-surface protein-expression measurements in conjunction with transcriptome-wide expression data.
  • the experiment is performed to confirm that the signal from the oligo on the antibody is reflective of cell- surface epitope concentration.
  • Experimental biases has been identified as coming from the following sources: 1) artificial signal arising from sequencing-library PCR duplicates, 2) cross-reactivity and availability of enough well-characterized antibody species, and 3) variable levels of oligo conjugation to the antibodies leading to inaccurate estimation of epitope concentration.
  • Methods of correcting the above identified biases include the following. First, PCR duplicates in sequencing datasets are filtered by the use of unique molecular identifiers (UMI) that are built into the design of the Drop-seq oligo. Second, to address antibody cross reactivity, the use of antibodies with low specificity is avoided and only highly optimized and tested flow-cytometry antibodies are used for benchmarking experiments. The optimized antibodies are available from large consortia such as the Human Protein Atlas which are continuously producing more antibodies to supplement the already existing pool of thousands of specific antibodies 7 . Third, the use of streptavidin-biotin conjugation, from the manufacturer's literature, it was estimated that roughly 4-12 oligonucleotide molecules are bound to each antibody molecule.
  • UMI unique molecular identifiers
  • Tagging one oligo molecule to each antibody makes the method at least as quantitative as Immuno-PCR-based approaches. For this purpose, evaluations are performed to test whether single-molecule signals can be confidently measured above noise in the final library quantification.
  • the optimal antibody concentration is also determined by titration experiments with individual antibodies as performed in flow cytometry. As a reference standard, the same monoclonal antibodies in a flow-cytometry run using the same cell populations is tested. This allows a determination of sensitivity and quantitative power of the sequencing-based ligonucleotide measurement.
  • Myeloid and lymphoid cell lineages have been extensively studied by cell-surface marker expression in flow cytometry and can be also identified based on their gene-expression profiles.
  • a number of well-established and highly specific flow-cytometry antibodies recognizing global markers for the myeloid and lymphoid lineages and for specific cell subpopulations within these lineages are linked to oligos described in Example 1.
  • the antibody-oligo complexes generated thereby are incubated with cells using conditions established for flow cytometry.
  • the cells are washed to remove unbound antibodies, then single cells are encapsulated into nanoliter-sized aqueous droplets in a microfluidic apparatus designed to perform Drop-seq 1 ( FIG. 1C ).
  • a microfluidic apparatus designed to perform Drop-seq 1 ( FIG. 1C ).
  • cell lysis which happens immediately in the droplets when the lysis buffer contacted cells
  • cellular mRNAs anneal to polyT containing Drop-seq beads ( FIG. 1B ) via their polyA tail.
  • Oligos from the antibody anneal to the Drop-seq beads via their poly-A stretch at the 3′ end.
  • a unique Barcode sequence on the Drop-seq bead indexes the transcriptome of each co-encapsulated cell. After breaking the emulsion and removing the oil, reverse transcription extends the Barcoded oligo to create the first-strand cDNA from both mRNA and antibody-derived oligo templates. The cDNA and the antibody-derived tags are separated by size, converted into Illumina-ready libraries and sequenced.
  • RNA-oligo complexes recognizing intracellular proteins are generated as described in Example 1. Furthermore, the established permeabilization and fixation procedure is performed before the incubation step of the CITE-seq protocol. Cells are identified based on protein expressed intracellularly and the mRNAs transcripts. This method not only provides a more detailed characterization of cell populations, but also allows studying post-transcriptional and post-translational gene regulation in single cells at an unprecedented depth.
  • Examples 1 to 5 are adapted to other droplet- or microwell-based single-cell sequencing technologies as described above.
  • the polyA stretch at the 3′end of the antibody-oligos allows capture in any oligo-dT-based mRNA-seq protocol, such as that described in Mortazavi et al 23 .
  • the run-specific parameters are evaluated and the utility of the method is assessed for commercially-available instruments (e.g., 10 ⁇ Genomics) and other technologies that are under development“.
  • tSNE t-distributed Stochastic Neighbor Embedding
  • FIG. 3B Another CITE-seq analysis of the same dataset of FIG. 3A was performed using RNA data alone. For example, CD8 and CD4 T cells were not separated into distinct populations. These results are shown in FIG. 3B , in which the dot plot demonstrates enhanced resolution when using multi-modal data.
  • the symbols in the figure are Mono (for monocytes), B for B cells, T for T cells, NK for natural killer cells, DC for conventional dendritic cells, pDC for plasmacytoid DC, Pre for precursors, and Ery for erythroblasts.
  • FIG. 4 shows bi-axial plots of CITE-seq antibody data for all 10 antibody constructs of Table 2. The data is comparable to what is obtained by flow cytometry with the significant difference that the transcriptome for every single cell (every dot) within these plots is also available when using CITE-seq. Cells can therefore be further analyzed and classified based on their RNA data, protein data, or both.
  • CITE-seq a method in which oligonucleotide-labeled antibodies are used to integrate cellular protein and transcriptome measurements into an efficient, single-cell readout.
  • CITE-seq is compatible with existing single-cell sequencing approaches and scales readily with throughput increases.
  • the CITE-seq method combines highly multiplexed protein marker detection with unbiased transcriptome profiling for thousands of single cells. The method is readily adaptable to two high-throughput scRNA-seq applications and shows that multimodal data analysis can achieve a more detailed characterization of cellular phenotypes than transcriptome measurements alone.
  • oligos oligonucleotides
  • oligo-dT primers used in most scRNA-seq library preparations
  • streptavidin-biotin interaction links the 5′ end of oligos to antibodies.
  • the antibody-oligo complexes are incubated with single-cell suspensions in conditions comparable to flow cytometry staining protocols; after this incubation, cells are washed to remove unbound antibodies and processed for scRNA-seq.
  • CITE-seq we asked whether quantitative differences in expression observed by flow cytometry can be observed by CITE-seq. For this, we focused on the marker CD8a, since its levels vary widely across immune cell populations.
  • CBMCs cord blood mononuclear cells
  • FACS fluorescence-activated cell sorting
  • the immune system has been extensively profiled using cell surface markers 47 and scRNA-see”, and both methods reliably identify the same cell types at consistent proportions.
  • a complex immune cell population is therefore an ideal system for validating the multimodal readout of CITE-seq.
  • CITE-seq panel of 13 well-characterized monoclonal antibodies that recognize cell-surface proteins routinely used as markers for immune-cell classification.
  • a rare spiked-in population of murine cells should be easily distinguished transcriptomically but should not cross-react with our anti-human antibodies; this would enable us to define background ADT levels directly from the data.
  • CITE-seq enables multimodal analysis of single cells at the scale afforded by droplet-based single-cell sequencing approaches.
  • oligo-bar-coded antibodies In contrast to flow and mass cytometry, detection of oligo-bar-coded antibodies is not limited by signal collision; a 10-nt sequence can easily encode more barcodes than there are human proteins, and this enables large-scale immunophenotyping with panels of tens to hundreds of antibodies.
  • mild cell permeabilization and fixation procedures used for intracellular cytometry assays should also be compatible with CITE- seq, and they may significantly expand the number of useful markers.
  • the CITE-seq readout was compared from different conjugation technologies.
  • One technology used the biotin-streptavidin (SAV) linkage previously described in Examples 1-8.
  • Another method for antibody-oligo conjugation employed covalent conjugation via iEDDA chemistry as described previously 45 .
  • the iEDDA conjugation chemistry used is comparable to conjugation chemistries offered in commercially available kits (Innova Biosciences, Thunderlink PLUS kit).
  • PBMCs peripheral blood mononuclear cells
  • tSNE t-distributed Stochastic Neighbor Embedding
  • the profiles (histograms) in different populations look comparable in the SAV and direct conjugation.
  • RNA-seq single cell RNA-seq
  • the demuxlet 71 algorithm enables the pooling of samples with distinct genotypes together into a single scRNA-seq experiment.
  • the sample-specific genetic polymorphisms serve as a fingerprint for the sample of origin, and therefore can be used to assign each cell to an individual after sequencing.
  • This workflow also enables the detection of multiplets originating from two individuals, reducing nonidentifiable multiplets at a rate that is directly proportional to the number of multiplexed samples. While this elegant approach requires pooled samples to originate from previously genotyped individuals, in principle any approach assigning sample fingerprints that can be measured alongside scRNA-seq would enable a similar strategy. For instance, sample multiplexing is frequently utilized in flow and mass cytometry by labeling distinct samples with antibodies to the same ubiquitously expressed surface protein, but conjugated to different fluorophores or isotopes, respectively 73,74 .
  • Cell hashtags allow for robust sample multiplexing, confident multiplet identification, and the discrimination of low-quality cells from ambient RNA.
  • this strategy represents a generalizable approach for doublet identification and multiplexing that can be tailored to any biological sample or experimental design.
  • HTO hashtag oligonucleotide
  • HTOs contain a different amplification handle than our standard CITE-seq antibody derived tags (ADT).
  • ADT CITE-seq antibody derived tags
  • HTOs, ADTs, and scRNA-seq libraries to be independently amplified and pooled at desired quantities.
  • PBMCs from eight separate human donors (referred to as donors A through H), and independently stained each sample with one of our HTO-conjugated antibody pools, while simultaneously performing a titration experiment with a pool of seven immunophenotypic markers for CITE-seq.
  • donors A through H we obtained PBMCs from eight separate human donors (referred to as donors A through H), and independently stained each sample with one of our HTO-conjugated antibody pools, while simultaneously performing a titration experiment with a pool of seven immunophenotypic markers for CITE-seq.
  • HTO signal between singlets we developed a straightforward statistical model to classify each barcode as “positive” or “negative” for each HTO. Briefly, we modeled the “background” signal for each HTO independently as a negative binomial distribution, estimating background cells based on the results of an initial k-medoids clustering of all HTO reads. Barcodes with HTO signals above the 99% quantile for this distribution were labeled as “positive”, and barcodes that were “positive” for more than one HTO were labeled as multiplets. We classified all barcodes where we detected at least 200 RNA UMI, regardless of HTO signal. Our classifications (visualized as a heatmap in FIG.
  • ‘negative’ barcodes did not separate based on their forced classification, consistent with these barcodes reflecting ambient RNA mixtures that may blend multiple subpopulations.
  • cell “hashing” can help recover low-quality cells that can otherwise be difficult to distinguish from ambient RNA ( FIG. 8F ).
  • PBMC genotyping Peripheral blood mononuclear cells were obtained from AllCells (USA). Genomic DNA was purified using the All-prep kit (Qiagen, USA) and genotyped using the Infinium core exome 24 array (Illumina, USA) according to manufacturer's instructions.
  • HEK293T human
  • NIH-3T3 mouse cells were maintained according to standard procedures in Dulbecco's Modified Eagle's Medium (Thermo Fisher, USA) supplemented with 10% fetal bovine serum (Thermo Fisher, USA) at 37° C. with 5% CO2.
  • Antibody-oligo conjugates directed against CD8 [clone: RPA-T8], CD45RA [clone: HI100], CD4 [clone: RPA-T4], HLA-DR [clone: L243], CD3 [clone: UCHT1], CCR7 [clone: G043H7] and PD-1 [clone: EH12.2H7] were provided by BioLegend (USA) containing 1-2 conjugated oligos per antibody on average.
  • Antibodies used for cell hashing were obtained as purified, unconjugated reagents from BioLegend (CD45 [clone: HI30], CD98 [clone: MEM-108], CD44 [clone: BJ18], and CD11a [clone: HI111]) and were covalently and irreversibly conjugated to HTOs by iEDDA-click chemistry as previously described 45 .
  • antibodies were washed into ix borate buffered saline (50 mM borate, 150 mM NaCl pH 8.5) and concentrated to 1 mg/ml using an Amicon Ultra 0.5 ml 30 kDa MWCO centrifugal filter (Millipore).
  • Methyltetrazine-PEG4-NHS ester (Click Chemistry Tools, USA) was dissolved in dry DMSO and added as a 30-fold excess to the antibody and allowed to react for 30 minutes at room temperature. Residual NHS groups were quenched by the addition of glycine and unreacted label was removed via centrifugal filtration. 5′-amine HTOs were ordered from Integrated DNA Technologies (USA) and reacted with a 20-fold excess of trans-cyclooctene-PEG4-NHS (Click Chemistry Tools, USA) in lx borate buffered saline supplemented with 20% DMSO for 30 minutes.
  • Residual NHS groups were quenched by the addition of glycine and residual label was removed by desalting (Bio-Rad Micro Bio-Spin P6).
  • Antibody-oligo conjugates were formed by mixing the appropriate labeled antibody and HTO and incubating at room temperature for at least 1 hour. Residual methyltetrazine groups on the antibody were quenched by the addition of trans-cyclooctene-PEG4-acid and unreacted oligo was removed centrifugal filtration using an Amicon Ultra 0.5 ml 50 kDa MWCO filter (Millipore, USA).
  • Antibody Titration Series To test optimal concentration of Antibody-Oligo conjugates provided by BioLegend (USA) per CITE-seq experiment, we tested 5 ⁇ g, 3 ⁇ g, 1 ⁇ g, 0.5 ⁇ g, 0.25 ⁇ g, 0.06 ⁇ g, and 0 ⁇ g for each conjugate. Titrations were staggered over the different batches to keep the total concentration of antibodies and oligos consistent between conditions (see Table 4 below).
  • PBMCs from different donors were independently stained with one of our HTO-conjugated antibody pools and a pool of 7 immunophenotypic markers for CITE-seq at different amounts (see above). All eight PBMC samples were pooled at equal concentration, alongside unlabeled HEK293T and mouse 3T3 as negative controls and loaded into the 10 ⁇ Chromium instrument (see Table 5 below).
  • CITE-seq on 10 ⁇ Genomics instrument Cells were “stained” with hashtagging antibodies and CITE-seq antibodies as described for CITE-see. “Stained” and washed cells were loaded into 10 ⁇ Genomics single cell 3′ v2 workflow and processed according to manufacturer's instructions up until the cDNA amplification step (10 ⁇ Genomics, USA). 2 pmol of HTO and ADT additive oligonucleotides were spiked into the cDNA amplification PCR and cDNA was amplified according to the 10 ⁇ Single Cell 3′ v2 protocol (10 ⁇ Genomics, USA).
  • RNA fraction derived from cellular mRNAs (retained on beads) from the ADT- and hashtag-containing fraction (in supernatant).
  • the cDNA fraction was processed according to the 10 ⁇ Genomics Single Cell 3′ v2 protocol to generate the transcriptome library.
  • An additional 1.4 ⁇ reaction volume of SPRI beads was added to the ADT/hashtag fraction to bring the ratio up to 2.0 ⁇ . Beads were washed with 80% ethanol, eluted in water, and an additional round of 2.0 ⁇ SPRI performed to remove excess single-stranded oligonucleotides from cDNA amplification.
  • VCF file that contained the individual genotype (GT) from the Infinium core exome 24 array output, using the PLINK command line tools (version 1.07).
  • This VCF file (which contained genotype information for the 8 PBMC donors as well as HEK cells), and the tagged ban) file from Drop-seq pipeline were used as inputs for demtmlet 71 , with default parameters.
  • RNA data were performed using the Seurat R package (version 2.1, Satija Lab) which enables the integrated processing of multi-modal (RNA, ADT, HTO) single cell datasets 78,79 .
  • Seurat R package version 2.1, Satija Lab
  • HEK identity As a separate test of HEK identity, we examined the demuxlet genotype for possible HEK cells. We observed 1,668 barcodes classified as HEK by the demtmlet algorithm, but whose transcriptomes clustered with PBMCs. These cells expressed ten-fold fewer UMI compared to transcriptomically-classified HEK cells, and did not express HEK-specific transcripts (i.e. NGFRAP1), both consistent with a PBMC identity. We therefore excluded these barcodes from all further analysis.
  • HTO raw counts were normalized using centered log ratio (CLR) transformation, where counts were divided by the geometric mean of an HTO across cells, and log-transformed:
  • xi denotes the count for a specified HTO in cell i
  • n is the total cell number
  • log denotes the natural log.
  • Barcodes that were positive for only one HTO were classified as singlets. Barcodes that were positive for two or more HTO were classified as doublets, and assigned sample IDs based on their two most highly expressed HTO. Barcodes that were negative for all eight HTO were classified as “negative”.
  • barcodes classified as “singlets” represent single cells, as we detect only a single HTO. However, they could also represent doublets of a PBMC with a HEK or 3T3 cell, as the latter two populations were unlabeled and represent negative controls. Indeed, when we analyzed the “HTO classification” of cells that were transcriptomically annotated as HEK or 3T3 cells, we found that 73.4% were annotated as “negative”, while 29.2% were annotated as singlets, in complete agreement with expected ratios in our “super-loaded” 10 ⁇ experiment. These cells appear in the heatmap in FIG. 6C , but all HEK and 3T3 cells were excluded from further analysis.
  • FIG. 1D For two-dimensional visualization of HTO levels ( FIG. 1D ), we used Euclidean distances calculated from the normalized HTO data as inputs for tSNE. Cells are colored based on their HTO classification as previously described.
  • FIG. 6F For visualization and clustering based on transcriptomic data ( FIG. 6F ), we first performed PCA on the 2,000 most highly variable genes (as determined by variance/mean ratio), and used the distance matrix defined by the first 11 principal components as input to tSNE and graph-based clustering in Seurat ( FIG. 6E ). We annotated the nine clusters based on canonical markers for known hematopoietic populations.
  • Demuxlet classifications were labeled as singlets (SNG), doublets (DBL) or ambiguous (AMB) according to the BEST column in the *.best output file.
  • SNG singlets
  • DBL doublets
  • AMB ambiguous
  • staining index based on standard approaches in flow cytometry, which examine the difference between positive and negative peak medians, divided by the spread (i.e. twice the mean absolute deviation) of the negative peak.
  • Discriminating low-quality cells from ambient RNA We performed HTO classification of low-quality barcodes (expressing between 50 and 200 UMI), using the previously determined HTO thresholds. For each barcode, we classified its expression as one of our previously determined nine hematopoietic populations using random forests, as implemented in the ranger package in R27. We first trained a classifier on the 13,757 PBMCs, using the 2,000 most variable genes as input, and their clustering identities as training labels. We then applied this classifier to each of the low-quality barcodes. We note that this classifier is guaranteed to return a result for each barcode.
  • Combinatorial split-pool hashtagging can be used to increase the number of barcodes and thereby increase doublet detection capability.
  • the Hashtagging approach is inherent in in-situ barcoding approaches (SPLiT-seq, sci-RNAseq) if the first round of barcoding defines different conditions or samples. In contrast to demuxlet, this approach can be used to multiplex samples of the same genotype. No need to perform genotyping on sample. This process can be extended to barcoding nuclei.
  • both cell “hashing” and genetic multiplexing allow the “super loading” of scRNA-seq platforms.
  • This benefit applies to any single-cell technology that relies on Poisson loading for cell isolation.
  • the per-cell cost savings for library preparation can therefore be significant, approaching an order of magnitude as the number of multiplexed samples increases.
  • cell “hashing” enables even a single sample to be highly multiplexed, as cells can be split into an arbitrary number of pools.
  • savings in library prep are partially offset by reads originating from multiplets, which must be sequenced and discarded.
  • multiplexing should facilitate the generation of large scRNA-seq and CITE-seq datasets.
  • Informatic detection of multiplets based on transcriptomic data also remains an important challenge for the field, for example, to identify doublets originating from two cells within the same sample.
  • cell or nucleus including other protein:protein interactions, aptamers 77 , or direct chemical conjugation of oligos to cells or nuclei.
  • n is a or g or c or t/u 5 ⁇ 223> Synthetic oligonucleotide sequence for CBMC profiling 6 ⁇ 223> Synthetic oligonucleotide sequence for CMBC profiling 7 ⁇ 223> Synthetic oligonucleotide sequence for CMBC profiling 8 ⁇ 223> Synthetic oligonucleotide sequence for CMBC profiling 9 ⁇ 223> Synthetic oligonucleotide sequence for CMBC profiling 10 ⁇ 223> Synthetic oligonucleotide sequence for CBMC profiling 11 ⁇ 223> Synthetic oligonucleotide sequence for CMBC profiling 12 ⁇ 223> Synthetic oligonucleotide sequence for CMBC profiling 13 ⁇ 223> Synthetic oligonucleotide sequence for CMBC profiling 14 ⁇ 223> Synthetic oligonucleotide sequence for CMBC profiling 15
  • n is A or C or T or G forming a barcode or index sequence ⁇ 220> ⁇ 221> misc_feature ⁇ 222> (53) . . . (54) ⁇ 223> Bases modified by phosphorothioate bond

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