Patent Grant
11732299
Cells within a tissue of a subject have differences in cell morphology and/or function due to varied analyte levels (e.g., gene and/or protein expression) within the different cells. The specific position of a cell within a tissue (e.g., the cell's position relative to neighboring cells or the cell's position relative to the tissue microenvironment) can affect, e.g., the cell's morphology, differentiation, fate, viability, proliferation, behavior, and signaling and cross-talk with other cells in the tissue.
Spatial heterogeneity has been previously studied using techniques that only provide data for a small handful of analytes in the context of an intact tissue or a portion of a tissue, or provide a lot of analyte data for single cells, but fail to provide information regarding the position of the single cell in a parent biological sample (e.g., tissue sample).
Genetic material, and related gene and protein expression, influences cellular fate and behavior. Screens such as those based on CRISPR can help elucidate gene function.
However, current methods have inherent limitations, and it remains difficult to assay complex phenotypes including transcriptional profiles.
This disclosure relates to methods for spatial profiling at least one biological analyte present in a cell comprising a small molecule.
In one aspect provided herein is a method for spatial profiling a biological analyte in a cell comprising: (a) contacting the cell with a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality comprises a first barcode and a capture domain, and wherein the cell comprises a small molecule and a second barcode; (b) releasing the biological analyte from the cell, wherein the biological analyte is bound by the capture probe at a distinct spatial position of the substrate; (c) detecting the biological analyte bound by the capture probe; and (e) correlating the biological analyte with the first barcode and the second barcode at the distinct spatial position of the substrate, thus profiling the biological analyte as present in the cell at the distinct spatial position. In some embodiments, the small molecule and second barcode are introduced into the cell using a particle.
In some instances, disclosed herein is a method for determining the presence or abundance of a moiety in a cell comprising: (a) contacting the cell with an array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a spatial barcode and (ii) a capture domain, wherein the cell comprises the moiety and wherein the moiety comprises a moiety sequence; (b) hybridizing a moiety sequence to the capture domain; and (c) determining (i) all or a part of the sequence of the moiety sequence bound to the capture domain, or a complement thereof, and (ii) all or a part of the sequence of the spatial barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to determine the presence or abundance of the moiety in the cell.
Also provided herein is a method for spatial profiling a biological analyte in a cell comprising: (a) contacting a plurality of cells with a plurality of particles, wherein a particle of the plurality of particles comprises a small molecule and a second barcode, and wherein the cell uptakes the small molecule and the second barcode; (b) contacting the cell with a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality comprises a first barcode and a capture domain; (c) releasing the biological analyte from the cell, wherein the biological analyte is bound by the capture probe at a distinct spatial position of the substrate; (d) detecting the biological analyte bound by the capture probe; and (e) correlating the biological analyte with the first barcode and the second barcode at the distinct spatial position of the substrate, thus profiling the biological analyte as present in the cell at the distinct spatial position.
In some instances, the method further comprises: (a) hybridizing an analyte to a second capture domain of a second probe, wherein the second probe comprises a second spatial barcode and the second capture domain, and wherein the second capture probe is in proximity to the capture probe that is hybridized to the moiety sequence; and (b) determining (i) all or a part of a sequence of an analyte bound to the second capture domain, or a complement thereof, and (ii) all or a part of the sequence of the second spatial barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to determine the abundance of the analyte in the cell.
In some instances, the moiety sequence is substantially complementary to the capture domain, optionally wherein the moiety sequence is a polyadenylated sequence. In some instances, the moiety further comprises a particle introduced into the cell, wherein the particle comprises a small molecule and the moiety sequence. In some instances, the cell is from a plurality of cells, and wherein the plurality of cells is contacted with a plurality of particles, and wherein the cell uptakes the particle.
In some embodiments, the particle is a nanoparticle. In some embodiments, the particle comprises gold, silica, polyethylene glycol (PEG)-poly(lactide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PE2000, silver, cadmium-selenide, poly(methylacrylic) acid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-cholesterol-1,2-distearoyl-sn-glycero-3-phosphocholine (POPG), poly(lactic-co-glycolic acid) (PLGA)-polyethylene glycol, or a combination thereof.
In some embodiments, the particle comprises a surface modification. In some embodiments, the surface modification comprises cysteine-cyan5, a cationic monolayer, a nucleic acid, poly(isobutylene-alt-maleic anhydride), a coating with fetal bovine serum (FBS), citrate, 5-aminovaleric acid, L-DOPA, melatonin, serotonin-HCl, MUS/OT, glutathione/glucose, polyethyleneimine, or a combination thereof.
In some embodiments, the small molecule binds to a biological target. In some embodiments, the biological target is a protein or a nucleic acid. In some embodiments, the protein is a kinase, a receptor, a channel, an enzyme, or a combination thereof. In some embodiments, the protein is a G protein-coupled receptor, a kinase, a protease, an esterase, a phosphatase, ligand-gated ion channel, a voltage-gated ion channel, or a nuclear receptor.
In some embodiments, the small molecule inhibits the biological target. In some embodiments, the small molecule activates the biological target. In some embodiments, the cell is a mammalian cell.
In one aspect, provided herein is a method for spatial profiling a biological analyte present in a genetically-perturbed cell comprising: (a) contacting the genetically-perturbed cell with a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality comprises a first barcode and a capture domain; (b) releasing the biological analyte from the genetically-perturbed cell, wherein the biological analyte is bound by a capture probe at a distinct spatial position of the substrate; (c) detecting the biological analyte bound by the capture probe; and (e) correlating the biological analyte with the first barcode from the capture probe at the distinct spatial position of the substrate; thus profiling the biological analyte as present in the genetically-perturbed cell at the distinct spatial position.
In some embodiments, the genetically-perturbed cell comprises a clustered regularly interspaced short palindromic repeats (CRISPR)-based genetic perturbation. In some embodiments, the genetically-perturbed cell comprises a second barcode that identifies the genetic perturbation of the cell.
In some embodiments, the step of correlating the biological analyte with the first barcode of the capture probe at the distinct spatial position of the substrate further comprises correlating the genetic perturbation of the cell to a distinct spatial position of the substrate using the second barcode that identifies the genetic perturbation of the cell.
In some embodiments, the genetically-perturbed cell comprises a polyadenylated nucleotide sequence that identifies the genetic perturbation. In some embodiments, the step of correlating the biological analyte with the first barcode of the capture probe at the distinct spatial position of the substrate further comprises correlating the genetic perturbation of the cell to a distinct spatial position of the substrate using the polyadenylated nucleotide sequence.
Also provided herein is a method for spatial profiling a biological analyte present in a genetically-perturbed cell comprising: (a) transducing a cell with a vector to form the genetically-perturbed cell; (b) contacting the genetically-perturbed cell with a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises a first barcode and a capture domain; (c) releasing the biological analyte from the genetically-perturbed cell, wherein the biological analyte is bound by the capture probe at a distinct spatial position of the substrate; (e) detecting the biological analyte bound by the capture probe; and (f) correlating the biological analyte with the first barcode of the capture probe at the distinct spatial position of the substrate, thus profiling the biological analyte as present in the genetically-perturbed cell at the distinct spatial position.
In some embodiments, the vector is a lentiviral vector. In some embodiments, the lentiviral vector is a CRISPR lentiviral vector. In some embodiments, the CRISPR lentiviral vector comprises a guide RNA (gRNA).
In some embodiments, the CRISPR lentiviral vector comprises a second barcode identifying the gRNA. In some embodiments, the gRNA is a single guide RNA (sgRNA). In some embodiments, the step of correlating the biological analyte with the first barcode of the capture probe at the distinct spatial position of the substrate further comprises correlating the genetic perturbation to a distinct spatial position of the substrate using a second barcode that identifies the genetic perturbation.
In some embodiments, the vector comprises a polyadenylated or an oligo (dT) nucleotide sequence. In some embodiments, correlating the biological analyte with the first barcode of the capture probe at the distinct spatial position of the substrate further comprises correlating the genetic perturbation to the distinct spatial position of the substrate using the polyadenylated or the oligo (dT) nucleotide sequence. In some embodiments, the genetically-perturbed cell is a mammalian cell.
In some embodiments, the step of releasing the biological analyte comprises permeabilizing the cell. In some embodiments, the method further comprises fixing the cell prior to the permeabilizing the cell. In some embodiments, the method further comprises staining the cell prior to the permeabilizing the cell. In some embodiments, the cell is stained after the fixing the cell. In some embodiments, the cell is fixed and permeabilized prior to releasing the biological analyte from the biological sample. In some embodiments, the permeabilizing the cell comprises electrophoresis. In some embodiments, the permeabilizing the cell comprises administering a permeabilization reagent.
In some embodiments, the step of releasing the biological analyte comprises permeabilizing the genetically-perturbed cell. In some embodiments, the method further comprises fixing the genetically-perturbed cell prior to permeabilizing the genetically-perturbed cell. In some embodiments, the method further comprises staining the genetically-perturbed cell prior to permeabilizing the genetically-perturbed cell. In some embodiments, the genetically-perturbed cell is stained after the genetically-perturbed cell is fixed. In some embodiments, the genetically-perturbed cell is fixed and permeabilized prior to the step of releasing the biological analyte from the biological sample.
In some embodiments, the step of permeabilizing comprises electrophoresis. In some embodiments, the step of permeabilizing comprises administering a permeabilization reagent.
In some embodiments, the method further comprises imaging the genetically-perturbed cell. In some embodiments, the imaging is performed prior to releasing the biological analyte from the genetically-perturbed cell. In some embodiments, the imaging is performed after releasing the biological analyte from the genetically-perturbed cell. In some embodiments, the imaging is used to determine the morphology of the genetically-perturbed cell.
In some instances, the cell is a mammalian cell. In some instances, the cell is permeabilized prior to hybridizing the analyte to the capture domain. In some instances, the methods further include staining the cell prior to permeabilizing the cell.
In some embodiments, the method further comprises imaging the cell. In some embodiments, the imaging is performed prior to releasing the biological analyte from the cell. In some embodiments, the imaging is performed after releasing the biological analyte from the cell. In some embodiments, the imaging is used to determine the morphology of the cell.
In some embodiments, the capture probe comprises a unique molecular identifier. In some embodiments, the capture probe comprises a cleavage domain. In some embodiments, the capture probe comprises a functional domain. In some embodiments, the functional domain is a primer sequence. In some embodiments, the capture probe comprises a capture domain. In some embodiments, the capture domain comprises a poly-dT sequence. In some embodiments, the capture domain is configured to hybridize to a poly-A tail of an mRNA.
All publications, patents, patent applications, and information available on the internet and mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, and items of information incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.
Where values are described in terms of ranges, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.
The term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise.
The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, comprising mixtures thereof. “A and/or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”.
Various embodiments of the features of this disclosure are described herein. However, it should be understood that such embodiments are provided merely by way of example, and numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of this disclosure.
The following drawings illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner. Like reference symbols in the drawings indicate like elements.
Pooled screens have the potential to exponentially improve throughput and reduce overall costs dramatically due to the massive parallelization achieved through pooling. However, most pooling strategies to date have primarily focused on pooled screens involving nucleic acids (e.g., gRNA or plasmids). Moreover, methods to perform pooled genetic perturbation screens with morphometric phenotypic readouts combining spatial information with sequencing do not exist. A pooled screening approach with morphometric readout and spatial analysis using barcoded arrays offers a significant improvement in overall throughput and cost of performing pooled small molecule perturbation screens as well as pooled genetic perturbation screens. Thus, provided herein are methods for profiling a biological analyte, e.g., any of the analytes described herein, in a cell including a moiety (e.g., a small molecule and/or a genetic perturbation introduced into the cell). Also provided herein are methods for determining the abundance of a moiety, e.g., any of the moieties described herein, in a cell. Also provided herein are methods for determining the location of a biological analyte, e.g., any of the analytes as described herein, and/or a moiety, e.g., any of the moieties described herein, in a biological sample.
Spatial analysis methodologies and compositions described herein can provide a vast amount of analyte and/or expression data for a variety of analytes within a biological sample at high spatial resolution, while retaining native spatial context. Spatial analysis methods and compositions can include, e.g., the use of a capture probe including a spatial barcode (e.g., a nucleic acid sequence that provides information as to the location or position of an analyte within a cell or a tissue sample (e.g., mammalian cell or a mammalian tissue sample) and a capture domain that is capable of binding to an analyte (e.g., a protein and/or a nucleic acid) produced by and/or present in a cell. Spatial analysis methods and compositions can also include the use of a capture probe having a capture domain that captures an intermediate agent for indirect detection of an analyte. For example, the intermediate agent can include a nucleic acid sequence (e.g., a barcode) associated with the intermediate agent. Detection of the intermediate agent is therefore indicative of the analyte in the cell or tissue sample.
Non-limiting aspects of spatial analysis methodologies and compositions are described in U.S. Pat. Nos. 10,774,374, 10,724,078, 10,480,022, 10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810, 9,593,365, 8,951,726, 8,604,182, 7,709,198, U.S. Patent Application Publication Nos. 2020/239946, 2020/080136, 2020/0277663, 2020/024641, 2019/330617, 2019/264268, 2020/256867, 2020/224244, 2019/194709, 2019/161796, 2019/085383, 2019/055594, 2018/216161, 2018/051322, 2018/0245142, 2017/241911, 2017/089811, 2017/067096, 2017/029875, 2017/0016053, 2016/108458, 2015/000854, 2013/171621, WO 2018/091676, WO 2020/176788, Rodrigues et al., Science 363(6434):1463-1467, 2019; Lee et al., Nat. Protoc. 10(3):442-458, 2015; Trejo et al., PLoS ONE 14(2):e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gao et al., BMC Biol. 15:50, 2017; and Gupta et al., Nature Biotechnol. 36:1197-1202, 2018; the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C, dated June 2020), and/or the Visium Spatial Tissue Optimization Reagent Kits User Guide (e.g., Rev C, dated July 2020), both of which are available at the 10× Genomics Support Documentation website, and can be used herein in any combination. Further non-limiting aspects of spatial analysis methodologies and compositions are described herein.
Some general terminology that may be used in this disclosure can be found in Section (I)(b) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Typically, a “barcode” is a label, or identifier, that conveys or is capable of conveying information (e.g., information about an analyte in a sample, a bead, and/or a capture probe). A barcode can be part of an analyte, or independent of an analyte. A barcode can be attached to an analyte. A particular barcode can be unique relative to other barcodes. For the purpose of this disclosure, an “analyte” can include any biological substance, structure, moiety, or component to be analyzed. The term “target” can similarly refer to an analyte of interest.
Analytes can be broadly classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes. Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral proteins (e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, viral spike, etc.), extracellular and intracellular proteins, antibodies, and antigen binding fragments. In some embodiments, the analyte(s) can be localized to subcellular location(s), including, for example, organelles, e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc. In some embodiments, analyte(s) can be peptides or proteins, including without limitation antibodies and enzymes. Additional examples of analytes can be found in Section (I)(c) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. In some embodiments, an analyte can be detected indirectly, such as through detection of an intermediate agent, for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein.
A “biological sample” is typically obtained from the subject for analysis using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and/or other biological material from the subject. In some embodiments, a biological sample can be a tissue section. In some embodiments, a biological sample can be a fixed and/or stained biological sample (e.g., a fixed and/or stained tissue section). Non-limiting examples of stains include histological stains (e.g., hematoxylin and/or eosin) and immunological stains (e.g., fluorescent stains). In some embodiments, a biological sample (e.g., a fixed and/or stained biological sample) can be imaged. Biological samples are also described in Section (I)(d) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
In some embodiments, a biological sample is permeabilized with one or more permeabilization reagents. For example, permeabilization of a biological sample can facilitate analyte capture. Exemplary permeabilization agents and conditions are described in Section (I)(d)(ii)(13) or the Exemplary Embodiments Section of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
Array-based spatial analysis methods involve the transfer of one or more analytes from a biological sample to an array of features on a substrate, where each feature is associated with a unique spatial location on the array. Subsequent analysis of the transferred analytes includes determining the identity of the analytes and the spatial location of the analytes within the biological sample. The spatial location of an analyte within the biological sample is determined based on the feature to which the analyte is bound (e.g., directly or indirectly) on the array, and the feature's relative spatial location within the array.
A “capture probe” refers to any molecule capable of capturing (directly or indirectly) and/or labelling an analyte (e.g., an analyte of interest) in a biological sample. In some embodiments, the capture probe is a nucleic acid or a polypeptide. In some embodiments, the capture probe includes a barcode (e.g., a spatial barcode and/or a unique molecular identifier (UMI)) and a capture domain). In some embodiments, a capture probe can include a cleavage domain and/or a functional domain (e.g., a primer-binding site, such as for next-generation sequencing (NGS)). See, e.g., Section (II)(b) (e.g., subsections (i)-(vi)) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Generation of capture probes can be achieved by any appropriate method, including those described in Section (II)(d)(ii) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
The functional sequences can generally be selected for compatibility with any of a variety of different sequencing systems, e.g., Ion Torrent Proton or PGM, Illumina sequencing instruments, PacBio, Oxford Nanopore, etc., and the requirements thereof. In some embodiments, functional sequences can be selected for compatibility with non-commercialized sequencing systems. Examples of such sequencing systems and techniques, for which suitable functional sequences can be used, include (but are not limited to) Ion Torrent Proton or PGM sequencing, Illumina sequencing, PacBio SMRT sequencing, and Oxford Nanopore sequencing. Further, in some embodiments, functional sequences can be selected for compatibility with other sequencing systems, including non-commercialized sequencing systems.
In some embodiments, the spatial barcode 105 and functional sequences 104 is common to all of the probes attached to a given feature. In some embodiments, the UMI sequence 106 of a capture probe attached to a given feature is different from the UMI sequence of a different capture probe attached to the given feature.
In some embodiments, more than one analyte type (e.g., nucleic acids and proteins) from a biological sample can be detected (e.g., simultaneously or sequentially) using any appropriate multiplexing technique, such as those described in Section (IV) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
In some embodiments, detection of one or more analytes (e.g., protein analytes) can be performed using one or more analyte capture agents. As used herein, an “analyte capture agent” refers to an agent that interacts with an analyte (e.g., an analyte in a biological sample) and with a capture probe (e.g., a capture probe attached to a substrate or a feature) to identify the analyte. In some embodiments, the analyte capture agent includes: (i) an analyte binding moiety (e.g., that binds to an analyte), for example, an antibody or antigen-binding fragment thereof; (ii) analyte binding moiety barcode; and (iii) an analyte capture sequence. As used herein, the term “analyte binding moiety barcode” refers to a barcode that is associated with or otherwise identifies the analyte binding moiety. As used herein, the term “analyte capture sequence” refers to a region or moiety configured to hybridize to, bind to, couple to, or otherwise interact with a capture domain of a capture probe. In some cases, an analyte binding moiety barcode (or portion thereof) may be able to be removed (e.g., cleaved) from the analyte capture agent.
Additional description of analyte capture agents can be found in Section (II)(b)(ix) of WO 2020/176788 and/or Section (II)(b)(viii) U.S. Patent Application Publication No. 2020/0277663.
There are at least two methods to associate a spatial barcode with one or more neighboring cells, such that the spatial barcode identifies the one or more cells, and/or contents of the one or more cells, as associated with a particular spatial location. One method is to promote analytes or analyte proxies (e.g., intermediate agents) out of a cell and towards a spatially-barcoded array (e.g., including spatially-barcoded capture probes). Another method is to cleave spatially-barcoded capture probes from an array and promote the spatially-barcoded capture probes towards and/or into or onto the biological sample.
In some cases, capture probes may be configured to prime, replicate, and consequently yield optionally barcoded extension products from a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent (e.g., a ligation product or an analyte capture agent), or a portion thereof), or derivatives thereof (see, e.g., Section (II)(b)(vii) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663 regarding extended capture probes). In some cases, capture probes may be configured to form ligation products with a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent, or portion thereof), thereby creating ligations products that serve as proxies for a template.
As used herein, an “extended capture probe” refers to a capture probe having additional nucleotides added to the terminus (e.g., 3′ or 5′ end) of the capture probe thereby extending the overall length of the capture probe. For example, an “extended 3′ end” indicates additional nucleotides were added to the most 3′ nucleotide of the capture probe to extend the length of the capture probe, for example, by polymerization reactions used to extend nucleic acid molecules including templated polymerization catalyzed by a polymerase (e.g., a DNA polymerase or a reverse transcriptase). In some embodiments, extending the capture probe includes adding to a 3′ end of a capture probe a nucleic acid sequence that is complementary to a nucleic acid sequence of an analyte or intermediate agent specifically bound to the capture domain of the capture probe. In some embodiments, the capture probe is extended using reverse transcription. In some embodiments, the capture probe is extended using one or more DNA polymerases. The extended capture probes include the sequence of the capture probe and the sequence of the spatial barcode of the capture probe.
In some embodiments, extended capture probes are amplified (e.g., in bulk solution or on the array) to yield quantities that are sufficient for downstream analysis, e.g., via DNA sequencing. In some embodiments, extended capture probes (e.g., DNA molecules) act as templates for an amplification reaction (e.g., a polymerase chain reaction).
Additional variants of spatial analysis methods, including in some embodiments, an imaging step, are described in Section (II)(a) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Analysis of captured analytes (and/or intermediate agents or portions thereof), for example, including sample removal, extension of capture probes, sequencing (e.g., of a cleaved extended capture probe and/or a cDNA molecule complementary to an extended capture probe), sequencing on the array (e.g., using, for example, in situ hybridization or in situ ligation approaches), temporal analysis, and/or proximity capture, is described in Section (II)(g) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Some quality control measures are described in Section (II)(h) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
Spatial information can provide information of biological and/or medical importance. For example, the methods and compositions described herein can allow for: identification of one or more biomarkers (e.g., diagnostic, prognostic, and/or for determination of efficacy of a treatment) of a disease or disorder; identification of a candidate drug target for treatment of a disease or disorder; identification (e.g., diagnosis) of a subject as having a disease or disorder; identification of stage and/or prognosis of a disease or disorder in a subject; identification of a subject as having an increased likelihood of developing a disease or disorder; monitoring of progression of a disease or disorder in a subject; determination of efficacy of a treatment of a disease or disorder in a subject; identification of a patient subpopulation for which a treatment is effective for a disease or disorder; modification of a treatment of a subject with a disease or disorder; selection of a subject for participation in a clinical trial; and/or selection of a treatment for a subject with a disease or disorder.
Spatial information can provide information of biological importance. For example, the methods and compositions described herein can allow for: identification of transcriptome and/or proteome expression profiles (e.g., in healthy and/or diseased tissue); identification of multiple analyte types in close proximity (e.g., nearest neighbor analysis); determination of up- and/or down-regulated genes and/or proteins in diseased tissue; characterization of tumor microenvironments; characterization of tumor immune responses; characterization of cells types and their co-localization in tissue; and identification of genetic variants within tissues (e.g., based on gene and/or protein expression profiles associated with specific disease or disorder biomarkers).
Typically, for spatial array-based methods, a substrate functions as a support for direct or indirect attachment of capture probes to features of the array. A “feature” is an entity that acts as a support or repository for various molecular entities used in spatial analysis. In some embodiments, some or all of the features in an array are functionalized for analyte capture. Exemplary substrates are described in Section (II)(c) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. Exemplary features and geometric attributes of an array can be found in Sections (II)(d)(i), (II)(d)(iii), and (II)(d)(iv) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
Generally, analytes and/or intermediate agents (or portions thereof) can be captured when contacting a biological sample with a substrate including capture probes (e.g., a substrate with capture probes embedded, spotted, printed, fabricated on the substrate, or a substrate with features (e.g., beads, wells) comprising capture probes). As used herein, “contact,” “contacted,” and/or “contacting,” a biological sample with a substrate refers to any contact (e.g., direct or indirect) such that capture probes can interact (e.g., bind covalently or non-covalently (e.g., hybridize)) with analytes from the biological sample. Capture can be achieved actively (e.g., using electrophoresis) or passively (e.g., using diffusion). Analyte capture is further described in Section (II)(e) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
In some cases, spatial analysis can be performed by attaching and/or introducing a molecule (e.g., a peptide, a lipid, or a nucleic acid molecule) having a barcode (e.g., a spatial barcode) to a biological sample (e.g., to a cell in a biological sample). In some embodiments, a plurality of molecules (e.g., a plurality of nucleic acid molecules) having a plurality of barcodes (e.g., a plurality of spatial barcodes) are introduced to a biological sample (e.g., to a plurality of cells in a biological sample) for use in spatial analysis. In some embodiments, after attaching and/or introducing a molecule having a barcode to a biological sample, the biological sample can be physically separated (e.g., dissociated) into single cells or cell groups for analysis. Some such methods of spatial analysis are described in Section (III) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
In some cases, spatial analysis can be performed by detecting multiple oligonucleotides that hybridize to an analyte. In some instances, for example, spatial analysis can be performed using RNA-templated ligation (RTL). Methods of RTL have been described previously. See, e.g., Credle et al., Nucleic Acids Res. 2017 Aug. 21; 45(14):e128. Typically, RTL includes hybridization of two oligonucleotides to adjacent sequences on an analyte (e.g., an RNA molecule, such as an mRNA molecule). In some instances, the oligonucleotides are DNA molecules. In some instances, one of the oligonucleotides includes at least two ribonucleic acid bases at the 3′ end and/or the other oligonucleotide includes a phosphorylated nucleotide at the 5′ end. In some instances, one of the two oligonucleotides includes a capture domain (e.g., a poly(A) sequence, a non-homopolymeric sequence). After hybridization to the analyte, a ligase (e.g., SplintR ligase) ligates the two oligonucleotides together, creating a ligation product. In some instances, the two oligonucleotides hybridize to sequences that are not adjacent to one another. For example, hybridization of the two oligonucleotides creates a gap between the hybridized oligonucleotides. In some instances, a polymerase (e.g., a DNA polymerase) can extend one of the oligonucleotides prior to ligation. After ligation, the ligation product is released from the analyte. In some instances, the ligation product is released using an endonuclease (e.g., RNAse H). The released ligation product can then be captured by capture probes (e.g., instead of direct capture of an analyte) on an array, optionally amplified, and sequenced, thus determining the location and optionally the abundance of the analyte in the biological sample.
During analysis of spatial information, sequence information for a spatial barcode associated with an analyte is obtained, and the sequence information can be used to provide information about the spatial distribution of the analyte in the biological sample. Various methods can be used to obtain the spatial information. In some embodiments, specific capture probes and the analytes they capture are associated with specific locations in an array of features on a substrate. For example, specific spatial barcodes can be associated with specific array locations prior to array fabrication, and the sequences of the spatial barcodes can be stored (e.g., in a database) along with specific array location information, so that each spatial barcode uniquely maps to a particular array location.
Alternatively, specific spatial barcodes can be deposited at predetermined locations in an array of features during fabrication such that at each location, only one type of spatial barcode is present so that spatial barcodes are uniquely associated with a single feature of the array. Where necessary, the arrays can be decoded using any of the methods described herein so that spatial barcodes are uniquely associated with array feature locations, and this mapping can be stored as described above.
When sequence information is obtained for capture probes and/or analytes during analysis of spatial information, the locations of the capture probes and/or analytes can be determined by referring to the stored information that uniquely associates each spatial barcode with an array feature location. In this manner, specific capture probes and captured analytes are associated with specific locations in the array of features. Each array feature location represents a position relative to a coordinate reference point (e.g., an array location, a fiducial marker) for the array. Accordingly, each feature location has an “address” or location in the coordinate space of the array.
Some exemplary spatial analysis workflows are described in the Exemplary Embodiments section of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. See, for example, the Exemplary embodiment starting with “In some non-limiting examples of the workflows described herein, the sample can be immersed . . . ” of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663. See also, e.g., the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C, dated June 2020), and/or the Visium Spatial Tissue Optimization Reagent Kits User Guide (e.g., Rev C, dated July 2020).
In some embodiments, spatial analysis can be performed using dedicated hardware and/or software, such as any of the systems described in Sections (II)(e)(ii) and/or (V) of WO 2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663, or any of one or more of the devices or methods described in Sections Control Slide for Imaging, Methods of Using Control Slides and Substrates for, Systems of Using Control Slides and Substrates for Imaging, and/or Sample and Array Alignment Devices and Methods, Informational labels of WO 2020/123320.
Suitable systems for performing spatial analysis can include components such as a chamber (e.g., a flow cell or sealable, fluid-tight chamber) for containing a biological sample. The biological sample can be mounted for example, in a biological sample holder. One or more fluid chambers can be connected to the chamber and/or the sample holder via fluid conduits, and fluids can be delivered into the chamber and/or sample holder via fluidic pumps, vacuum sources, or other devices coupled to the fluid conduits that create a pressure gradient to drive fluid flow. One or more valves can also be connected to fluid conduits to regulate the flow of reagents from reservoirs to the chamber and/or sample holder.
The systems can optionally include a control unit that includes one or more electronic processors, an input interface, an output interface (such as a display), and a storage unit (e.g., a solid state storage medium such as, but not limited to, a magnetic, optical, or other solid state, persistent, writeable and/or re-writeable storage medium). The control unit can optionally be connected to one or more remote devices via a network. The control unit (and components thereof) can generally perform any of the steps and functions described herein. Where the system is connected to a remote device, the remote device (or devices) can perform any of the steps or features described herein. The systems can optionally include one or more detectors (e.g., CCD, CMOS) used to capture images. The systems can also optionally include one or more light sources (e.g., LED-based, diode-based, lasers) for illuminating a sample, a substrate with features, analytes from a biological sample captured on a substrate, and various control and calibration media.
The systems can optionally include software instructions encoded and/or implemented in one or more of tangible storage media and hardware components such as application specific integrated circuits. The software instructions, when executed by a control unit (and in particular, an electronic processor) or an integrated circuit, can cause the control unit, integrated circuit, or other component executing the software instructions to perform any of the method steps or functions described herein.
In some cases, the systems described herein can detect (e.g., register an image) the biological sample on the array. Exemplary methods to detect the biological sample on an array are described in PCT Application No. 2020/061064 and/or U.S. patent application Ser. No. 16/951,854.
Prior to transferring analytes from the biological sample to the array of features on the substrate, the biological sample can be aligned with the array. Alignment of a biological sample and an array of features including capture probes can facilitate spatial analysis, which can be used to detect differences in analyte presence and/or level within different positions in the biological sample, for example, to generate a three-dimensional map of the analyte presence and/or level. Exemplary methods to generate a two- and/or three-dimensional map of the analyte presence and/or level are described in PCT Application No. 2020/053655 and spatial analysis methods are generally described in WO 2020/061108 and/or U.S. patent application Ser. No. 16/951,864.
In some cases, a map of analyte presence and/or level can be aligned to an image of a biological sample using one or more fiducial markers, e.g., objects placed in the field of view of an imaging system which appear in the image produced, as described in the Substrate Attributes Section, Control Slide for Imaging Section of WO 2020/123320, PCT Application No. 2020/061066, and/or U.S. patent application Ser. No. 16/951,843. Fiducial markers can be used as a point of reference or measurement scale for alignment (e.g., to align a sample and an array, to align two substrates, to determine a location of a sample or array on a substrate relative to a fiducial marker) and/or for quantitative measurements of sizes and/or distances.
I. Spatial Analytical Methodology and Perturbation of Cells
(a) Introduction
A pooled screening approach that utilizes a morphometric readout and spatial analysis offers significant improvements for pooled perturbation screens. In such screens, a biological sample or a cell, e.g., a cell in a biological sample, can be perturbed by a perturbation agent. As described herein, a “perturbation agent” or “perturbation reagent” or “moiety” can be a small molecule, an antibody, a drug, an aptamer, a nucleic acid (e.g., miRNA), a CRISPR crRNA/sgRNA, TALEN, zinc finger nuclease, antisense oligonucleotide a physical environmental (e.g., temperature change), and/or any other known perturbation agents where the agent alters equilibrium or homeostasis. After perturbation of the biological sample or cell, e.g., a cell in a biological sample, the biological sample or cell comprising a moiety can be (i) imaged; and/or (ii) contacted with a spatial array to allow for profiling a biological analyte and/or determining the identity of the moiety in a cell at a distinct location within a biological sample. Such methods can be useful for determining the abundance of a biological analyte in a cell comprising a moiety at a distinct spatial position on a substrate. These methods can also be useful, for example, for detecting a perturbation (e.g., a change in a biological analyte such as a change in the amount of the biological analyte) in a cell comprising a moiety (e.g., a small molecule or genetic perturbation) compared to a cell comprising a different moiety or a cell not comprising a moiety.
Accordingly, provided herein are methods for determining the presence or abundance of a moiety in a cell comprising: (a) contacting the cell with an array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a spatial barcode and (ii) a capture domain, wherein the cell comprises the moiety and wherein the moiety comprises a moiety sequence; (b) hybridizing a moiety sequence to the capture domain; and (c) determining (i) all or a part of the sequence of the moiety sequence bound to the capture domain, or a complement thereof, and (ii) all or a part of the sequence of the spatial barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to determine the abundance of the moiety in the cell. In some embodiments, the method further includes determining (i) all or a part of a sequence of an analyte bound to a capture domain, or a complement thereof, and (ii) all or a part of the sequence of the spatial barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to determine the abundance of the analyte in the cell.
In some embodiments, provided herein are methods for determining an abundance of an analyte or moiety in a cell comprising: (a) contacting the cell with an array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a spatial barcode and (ii) a capture domain; wherein the cell comprises the moiety and wherein the moiety comprises a moiety sequence; (b) hybridizing the analyte or the moiety sequence to the capture domain; and (c) determining (i) all or a part of the sequence of the analyte or the moiety sequence bound to the capture domain, or a complement thereof, and (ii) all or a part of the sequence of the spatial barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to determine the abundance of the analyte or the moiety in the cell. A “moiety sequence” as used herein refers to a sequence that can be used to identify a moiety. For example, a moiety sequence can be unique to each moiety (e.g., each small molecule or each genetic perturbation).
In some embodiments, the methods provided herein include profiling a biological analyte, e.g., any of the analytes as described herein, in a cell including a moiety (e.g., a small molecule and/or genetic perturbation introduced into the cell). Also provided herein are methods for determining the abundance of a moiety, e.g., any of the moieties described herein, in a cell. In some embodiments, the cell is a member of a plurality of cells, e.g., the cell is in a biological sample. In some embodiments, the methods provided herein include determining the location of a biological analyte, e.g., any of the analytes as described herein, and/or a moiety, e.g., any of the moieties described herein, in a biological sample. In some instances, both the abundance and the location of a biological analyte and/or a moiety are determined.
Also provided herein are methods for profiling a biological analyte, e.g., any of the analytes as described herein, in a cell including a small molecule (e.g., a small molecule introduced into the cell). In some embodiments, the methods described herein can include releasing a biological analyte from a cell including a small molecule (e.g., a small molecule introduced into the cell). The released biological analyte can be bound by a capture probe as described herein at a distinct spatial position on a substrate and detected. The bound biological analyte can then be correlated with a barcode of the capture probe at a distinct spatial position of the substrate.
Also provided herein is a method for spatial profiling a biological analyte in a cell including: contacting a plurality of cells with a plurality of particles, wherein a particle of the plurality of particles includes a small molecule and a second barcode, and wherein the cell uptakes the small molecule and the second barcode; contacting the cell with a substrate including a plurality of capture probes, wherein a capture probe of the plurality includes a first barcode and a capture domain; releasing the biological analyte from the cell, wherein the biological analyte is bound by the capture probe at a distinct spatial position of the substrate; detecting the biological analyte bound by the capture probe; and correlating the biological analyte with the first barcode and the second barcode at the distinct spatial position of the substrate, thus profiling the biological analyte as present in the cell at the distinct spatial position.
In some embodiments, a method for spatial profiling a biological analyte in a cell includes: contacting the cell with a substrate including a plurality of capture probes, wherein a capture probe of the plurality includes a first barcode and a capture domain, and wherein the cell includes a small molecule and a second barcode; releasing the biological analyte from the cell, wherein the biological analyte is bound by the capture probe at a distinct spatial position of the substrate; detecting the biological analyte bound by the capture probe; and correlating the biological analyte with the first barcode and the second barcode at the distinct spatial position of the substrate, thus profiling the biological analyte as present in the cell at the distinct spatial position.
In some embodiments, the methods described herein can include releasing a biological analyte from a genetically-perturbed cell. The released biological analyte can be bound by a capture probe as described herein at a distinct spatial position on a substrate and detected. The bound biological analyte can then be correlated with a barcode of the capture probe at a distinct spatial position of the substrate. Such methods can be useful for correlating a genetic perturbation of a cell to a biological analyte at a distinct spatial position on a substrate.
Also provided herein are methods for spatial profiling a biological analyte present in a genetically-perturbed cell that include transducing a cell with a vector to form the genetically-perturbed cell; contacting the genetically-perturbed cell with a substrate including a plurality of capture probes, wherein a capture probe of the plurality of capture probes includes a first barcode and a capture domain; releasing the biological analyte from the genetically-perturbed cell, wherein the biological analyte is bound by the capture probe at a distinct spatial position of the substrate; detecting the biological analyte bound by the capture probe; and correlating the biological analyte with the first barcode of the capture probe at the distinct spatial position of the substrate, thus profiling the biological analyte as present in the genetically-perturbed cell at the distinct spatial position.
In some embodiments, a method for spatially profiling a biological analyte present in a genetically-perturbed cell includes contacting the genetically-perturbed cell with a substrate including a plurality of capture probes, wherein a capture probe of the plurality includes a first barcode and a capture domain; releasing the biological analyte from the genetically-perturbed cell, wherein the biological analyte is bound by a capture probe at a distinct spatial position of the substrate; detecting the biological analyte bound by the capture probe; and correlating the biological analyte with the first barcode from the capture probe at the distinct spatial position of the substrate; thus profiling the biological analyte as present in the genetically-perturbed cell at the distinct spatial position.
(b) Generation of Libraries
1. Generating Pooled Small Molecule Libraries
In some embodiments, disclosed herein are methods of generating a small molecule library. Methods of generating a small molecule library have been described, for example, in U.S. Pat. Nos. 8,951,728; 6,677,160; Dandapani et al. Curr Protoc Chem Biol. 4:177-191, 2012; Hajduk et al. Nature. 470:42-43, 2011; Paricharak et al. Briefings in Bioinformatics. 19(2):277-285, 2018; and Harris et al. Comb Chem High Throughput Screen. 14(6):521-531, 2011; each of which is incorporated herein by reference in its entirety. Generating a barcoded small molecule library can include loading a plurality of particles with a plurality of small molecules and a plurality of moiety sequences (e.g., a second barcode). In some embodiments, a moiety sequence (e.g., a second barcode) is affixed to each small molecule.
Methods of introducing a small molecule into a cell are known to one of skill in art. Such methods include using particles (e.g., nanoparticles) to introduce the small molecule into the cell. Accordingly, in some embodiments, a moiety as described herein further comprises a particle introduced into the cell. In some embodiments, the particle is taken up by the cell through diffusion, electroporation, receptor-mediated endocytosis, or a combination thereof. See, e.g., Behzadi et al., Chem Soc Rev. 46(14): 4218-4244, 2017; Mosquera et al., Acc Chem Res, 51(9):2305-2313, 2018; Jahangirian et al., Int J Nanomedicine, 14:1633-1657, 2019; and Zhao, Scientific Reports. 7:4131, 2017; each of which is incorporated herein by reference in its entirety. In some embodiments, the cell is from a plurality of cells, and the plurality of cells is contacted with a plurality of particles, and a cell uptakes the particle. In some embodiments, the particle includes a moiety sequence. In some embodiments, the moiety sequence is substantially complementary to the capture domain. In some embodiments, the moiety sequence is a polyadenylated sequence.
In some embodiments, the small molecule library includes small molecules with validated biological and pharmacological activities with particular solubility, purity, and stability of the compounds. In some embodiments, the library is fully randomized, with no sequence preferences or constants at any position. In another embodiment, the library is biased.
In some embodiments, the small molecule binds to a biological target. In some embodiments, the biological target is a protein or a nucleic acid. In some embodiments, the protein is a kinase, a receptor, a channel, an enzyme, or a combination thereof. In some embodiments, the protein is a G protein-coupled receptor, a kinase, a protease, an esterase, a phosphatase, ligand-gated ion channel, a voltage-gated ion channel, or a nuclear receptor. In some embodiments, the small molecule interacts with a known cellular molecule or known classes of cellular molecules. In some embodiments, the small molecules include but are not limited to inhibitors, antagonists, and agonists of various cellular pathways, including for example, pathways involving DNA damage/DNA repair, cell cycle/checkpoints, JAK/STAT signaling, MAPK signaling, GPCR/G protein, angiogenesis, immunology and inflammation, endocrinology and hormones, cancer, metabolism, and stem cells. In some embodiments, the small molecule inhibits the biological target. In some embodiments, the small molecule activates the biological target. In some embodiments, correlating the biological analyte with the first barcode and the second barcode at the distinct spatial position of the substrate, thus profiling the biological analyte as present in the cell at the distinct spatial position.
In some embodiments, one or more small molecules are encapsulated into one or more particles. In some embodiments, one or more small molecules and one or more moiety sequences (e.g., second barcodes) are encapsulated into one or more particles. In some embodiments, one or more small molecules are loaded onto one or more particles. Any particle that can introduce a small molecule into a cell can be used. Such particles can include, for example, gold, silica, polyethylene glycol (PEG)-poly(lactide), silver, cadmium-selenide, poly(methylacrylic) acid, a lipid (e.g., 2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-cholesterol-1,2-distearoyl-sn-glycero-3-phosphocholine (POPG) and poly(lactic-co-glycolic acid) (PLGA)-polyethylene glycol), polystyrene, carboxylated polystyrene, NH2-labeled polystyrene, polystyrene latex, fullerene, alginate-chitosan, a polymer-lipid hybrid (e.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PE2000), a quantum dot, and any combination thereof.
In some embodiments, a particle described herein can be spherical or disc-shaped. In some embodiments, the diameter of the particle is about 1 nm to about 2500 nm. For example, about 1 nm to about 200 nm, about 1 nm to about 400 nm, about 1 nm to about 600 nm, about 1 nm to about 800 nm, about 1 nm to about 1000 nm, about 1 nm to about 1200 nm, about 1 nm to about 1400 nm, about 1 nm to about 1600 nm, about 1 nm to about 1800 nm, about 1 nm to about 2000 nm, about 1 nm to about 2200 nm, about 1 nm to about 2400 nm, about 2300 nm to about 2500 nm, about 2100 nm to about 2500 nm, about 1900 nm to about 2500 nm, about 1700 nm to about 2500 nm, about 1500 nm to about 2500 nm, about 1300 nm to about 2500 nm, about 1100 nm to about 2500 nm, about 900 nm to about 2500 nm, about 700 nm to about 2500 nm, about 500 nm to about 2500 nm, about 300 nm to about 2500 nm, or about 100 nm to about 2500 nm. In some embodiments, the diameter of the particle is about 5 nm to about 500 nm. For example, about 5 nm to about 50 nm, about 5 nm to about 100 nm, about 5 nm to about 150 nm, about 5 nm to about 200 nm, about 5 nm to about 250 nm, about 5 nm to about 300 nm, about 5 nm to about 350 nm, about 5 nm to about 400 nm, about 5 nm to about 450 nm, about 450 nm to about 500 nm, about 400 nm to about 500 nm, about 350 nm to about 500 nm, about 300 nm to about 500 nm, about 250 nm to about 500 nm, about 200 nm to about 500, about 150 nm to about 500 nm, about 100 nm to about 500 nm, or about 50 nm to about 500 nm. For example, about 5 nm to about 25 nm, about 25 nm to about 50 nm, about 40 nm to about 60 nm, about 50 to about 75 nm, or about 75 nm to about 100 nm.
In some embodiments, the particle is rod-shaped. As used herein, a “rod-shaped particle” can also refer to a rice-like particle, a worm-like particle, and a cylindrical particle. In some embodiments, the length of the particle is about 1 nm to about 2500 nm. For example, about 1 nm to about 200 nm, about 1 nm to about 400 nm, about 1 nm to about 600 nm, about 1 nm to about 800 nm, about 1 nm to about 1000 nm, about 1 nm to about 1200 nm, about 1 nm to about 1400 nm, about 1 nm to about 1600 nm, about 1 nm to about 1800 nm, about 1 nm to about 2000 nm, about 1 nm to about 2200 nm, about 1 nm to about 2400 nm, about 2300 nm to about 2500 nm, about 2100 nm to about 2500 nm, about 1900 nm to about 2500 nm, about 1700 nm to about 2500 nm, about 1500 nm to about 2500 nm, about 1300 nm to about 2500 nm, about 1100 nm to about 2500 nm, about 900 nm to about 2500 nm, about 700 nm to about 2500 nm, about 500 nm to about 2500 nm, about 300 nm to about 2500 nm, or about 100 nm to about 2500 nm. In some embodiments, the length of the particle is about 5 nm to about 500 nm. For example, about 5 nm to about 50 nm, about 5 nm to about 100 nm, about 5 nm to about 150 nm, about 5 nm to about 200 nm, about 5 nm to about 250 nm, about 5 nm to about 300 nm, about 5 nm to about 350 nm, about 5 nm to about 400 nm, about 5 nm to about 450 nm, about 450 nm to about 500 nm, about 400 nm to about 500 nm, about 350 nm to about 500 nm, about 300 nm to about 500 nm, about 250 nm to about 500 nm, about 200 nm to about 500, about 150 nm to about 500 nm, about 100 nm to about 500 nm, or about 50 nm to about 500 nm. For example, about 5 nm to about 25 nm, about 25 nm to about 50 nm, about 40 nm to about 60 nm, about 50 to about 75 nm, or about 75 nm to about 100 nm. In some embodiments, the rod-shaped particle has an aspect ratio (i.e., the ratio of length to width) of about 1 to about 8. For example an aspect ratio of about 1 to about 2, about 1 to about 3, about 1 to about 4, about 1 to about 5, about 1 to about 6, about 1 to about 7, about 7 to about 8, about 6 to about 8, about 5 to about 8, about 4 to about 8, about 3 to about 8, or about 2 to about 8. In some embodiments, the rod-shaped particle has an aspect ratio of about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, or about 7.5.
In some embodiments, the particle is hydrophilic. In some embodiments, the particle is hydrophobic. In some embodiment, the particle is cationic. In some embodiments, the particle has a surface charge of about 1 to about 40 mV. In some embodiments, the particle is anionic. In some embodiments, the particle has a surface charge of about −1 to about −70 mV.
In some embodiments, the particle is thermo-responsive. For example, the hydrophobicity of a thermo-responsive particle can be controlled by temperature. The particles can also include surface modifications. Non-limiting examples of such surface modifications include cysteine-cyan5, a cationic monolayer, a nucleic acid, poly(isobutylene-alt-maleic anhydride), a coating with fetal bovine serum (FBS), citrate, 5-aminovaleric acid, L-DOPA, melatonin, serotonin-HCl, MUS/OT, glutathione/glucose, polyethyleneimine, or a combination thereof. See, e.g., Donahue. Adv Drug Deliv Rev. 143:68-96, 2019, which is incorporated herein by reference in its entirety.
In some instances, the small molecule can include a moiety sequence (e.g., a second barcode) (e.g., an oligonucleotide). In some instances, the moiety sequence (e.g., a second barcode) comprises a sequence that is unique to the small molecule (e.g., a unique molecular identifier (UMI) for the small molecule). In some instances, this UMI can be used to identify the presence of the small molecule in a biological sample (i.e., a cell). In some instances, the moiety sequence (e.g., a second barcode) further comprises a sequence that can hybridize to at least a portion or an entirety of a capture domain of a capture probe. In some embodiments, the small molecule includes a moiety sequence (e.g., a second barcode) that is conjugated or otherwise attached to the small molecule. In some embodiments, the moiety sequence (e.g., a second barcode) is covalently-linked to the small molecule. In some embodiments, a moiety sequence (e.g., a second barcode) is a nucleic acid sequence.
As used herein, the term “moiety sequence” (or “second barcode”) refers to a barcode that is associated with or otherwise identifies the small molecule. In some embodiments, by identifying a small molecule and its associated moiety sequence, the analyte to which the small molecule binds can also be identified. A moiety sequence can be a nucleic acid sequence of a given length and/or sequence that is associated with the small molecule. A moiety sequence can generally include any of the variety of aspects of barcodes described herein.
In some embodiments, the moiety sequence comprises a region or moiety configured to hybridize to, bind to, couple to, or otherwise interact with a capture domain of a capture probe. In some embodiments, a moiety sequence includes a nucleic acid sequence that is complementary to or substantially complementary to the capture domain of a capture probe such that the moiety sequence hybridizes to the capture domain of the capture probe. In some embodiments, a moiety sequence comprises a poly(A) nucleic acid sequence that hybridizes to a capture domain that comprises a poly(T) nucleic acid sequence. In some embodiments, a moiety sequence comprises a poly(T) nucleic acid sequence that hybridizes to a capture domain that comprises a poly(A) nucleic acid sequence. In some embodiments, a moiety sequence comprises a non-homopolymeric nucleic acid sequence that hybridizes to a capture domain that comprises a non-homopolymeric nucleic acid sequence that is complementary (or substantially complementary) to the non-homopolymeric nucleic acid sequence of the moiety sequence.
In some embodiments of any of the spatial analysis methods described herein, the moiety sequence can be directly coupled to the small molecule, or it can be attached to a bead, molecular lattice, e.g., a linear, globular, cross-slinked, or other polymer, or other framework that is attached or otherwise associated with the small molecule, which allows attachment of multiple moiety sequences to a single small molecule. Attachment (coupling) of the moiety sequences to the small molecule can be achieved through any of a variety of direct or indirect, covalent or non-covalent associations or attachments. For example, in the case of a moiety sequence coupled to a small molecule that includes an antibody or antigen-binding fragment, such a moiety sequence can be covalently attached to a portion of the antibody or antigen-binding fragment using chemical conjugation techniques (e.g., Lightning-Link® antibody labelling kits available from Innova Biosciences). In some embodiments, a moiety sequence can be coupled to an antibody or antigen-binding fragment using non-covalent attachment mechanisms (e.g., using biotinylated antibodies and oligonucleotides or beads that include one or more biotinylated linker(s), coupled to oligonucleotides with an avidin or streptavidin linker.) Antibody and oligonucleotide biotinylation techniques can be used, and are described for example in Fang et al., Nucleic Acids Res. (2003), 31(2): 708-715, the entire contents of which is incorporated by reference herein. Likewise, protein and peptide biotinylation techniques have been developed and can be used, and are described for example in U.S. Pat. No. 6,265,552, the entire contents of which is incorporated by reference herein. Furthermore, click reaction chemistry such as a methyltetrazine-PEG5-NHS ester reaction, a TCO-PEG4-NHS ester reaction, or the like, can be used to couple moiety sequences to small molecules. The reactive moiety on the small molecule can also include amine for targeting aldehydes, amine for targeting maleimide (e.g., free thiols), azide for targeting click chemistry compounds (e.g., alkynes), biotin for targeting streptavidin, phosphates for targeting EDC, which in turn targets active ester (e.g., NH2). Exemplary strategies to conjugate the small molecule to the moiety sequence include the use of commercial kits (e.g., Solulink, Thunder link), conjugation of mild reduction of hinge region and maleimide labelling, stain-promoted click chemistry reaction to labeled amides (e.g., copper-free), and conjugation of periodate oxidation of sugar chain and amine conjugation.
In some embodiments of any of the spatial profiling methods described herein, the moiety sequence coupled to a small molecule can include modifications that render it non-extendable by a polymerase. In some embodiments, when binding to a capture domain of a capture probe or nucleic acid in a sample for a primer extension reaction, the moiety sequence can serve as a template, not a primer. In some embodiments, the moiety sequence can include a random N-mer sequence that is capped with modifications that render it non-extendable by a polymerase. In some cases, the composition of the random N-mer sequence can be designed to maximize the binding efficiency to free, unbarcoded ssDNA molecules. The design can include a random sequence composition with a higher GC content, a partial random sequence with fixed G or C at specific positions, the use of guanosines, the use of locked nucleic acids, or any combination thereof.
A modification for blocking primer extension by a polymerase can be a carbon spacer group of different lengths or a dideoxynucleotide. In some embodiments, the modification can be an abasic site that has an apurine or apyrimidine structure, a base analog, or an analogue of a phosphate backbone, such as a backbone of N-(2-aminoethyl)-glycine linked by amide bonds, tetrahydrofuran, or 1′, 2′-Dideoxyribose. The modification can also be a uracil base, 2′OMe modified RNA, C3-18 spacers (e.g., structures with 3-18 consecutive carbon atoms, such as C3 spacer), ethylene glycol multimer spacers (e.g., spacer 18 (hexa-ethyleneglycol spacer), biotin, di-deoxynucleotide triphosphate, ethylene glycol, amine, or phosphate.
In some embodiments of any of the spatial profiling methods described herein, the moiety sequence includes a cleavable domain. For example, after the small molecule binds to an analyte (e.g., a cell surface analyte), the moiety sequence can be cleaved and collected for downstream analysis according to the methods as described herein. In some embodiments, the cleavable domain of the moiety sequence includes a U-excising element that allows the species to release from the bead. In some embodiments, the U-excising element can include a single-stranded DNA (ssDNA) sequence that contains at least one uracil. The species can be attached to a bead via the ssDNA sequence. The species can be released by a combination of uracil-DNA glycosylase (e.g., to remove the uracil) and an endonuclease (e.g., to induce an ssDNA break). If the endonuclease generates a 5′ phosphate group from the cleavage, then additional enzyme treatment can be included in downstream processing to eliminate the phosphate group, e.g., prior to ligation of additional sequencing handle elements, e.g., Illumina full P5 sequence, partial P5 sequence, full R1 sequence, and/or partial R1 sequence.
In some embodiments, multiple different species of analytes (e.g., polypeptides) from the biological sample can be subsequently associated with the one or more physical properties of the biological sample. For example, the multiple different species of analytes can be associated with locations of the analytes in the biological sample. Such information (e.g., proteomic information when the small molecule(s) recognizes a polypeptide(s)) can be used in association with other spatial information (e.g., genetic information from the biological sample, such as DNA sequence information, transcriptome information (i.e., sequences of transcripts), or both). For example, a cell surface protein of a cell can be associated with one or more physical properties of the cell (e.g., a shape, size, activity, or a type of the cell). The one or more physical properties can be characterized by imaging the cell.
2. Generating Pooled Nucleic Acid Libraries
Also provided herein are methods of generating a library of genetically-perturbed cells. A genetically-perturbed cell can refer to any cell that has one or more genetic mutations including, but not limited to, a nucleotide deletion, insertion, or substitution. A cell comprising a moiety can be a genetically-perturbed cell. In some embodiments, a genetically-perturbed cell (i.e., a cell comprising a moiety) can refer to a cell that has a gene knockout and/or a gene knockdown. Methods of introducing a genetic perturbation into a cell and methods for generating a library of genetically-perturbed cells are known to one of skill in art. Such methods have been described in, for example, Liberali et al. Nat Rev Genet. 2015; and 16(1):18-32; and Boutros and Ahringer. Nat Rev Genet. 2008; 9(7):554-66.
In some embodiments, a cell comprising a moiety has been transduced with a vector, e.g., any of the vectors described herein. In some embodiments, a “genetically-perturbed cell” refers to a cell that has been transduced with a vector. In some embodiments, methods of introducing a genetic perturbation or moiety into a cell and/or methods for generating a library of genetically-perturbed cells include using a vector. In some embodiments, the vector is not integrated into the host cell's genome. In some embodiments, the vector is integrated into the host cell's genome.
Non-limiting examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vectors (e.g., pSV or pCMV vectors), adeno-associated virus (AAV) vectors, lentivirus vectors, and retroviral vectors), and any Gateway® vectors. A vector can, for example, include sufficient cis-acting elements for expression where other elements for expression can be supplied by the host mammalian cell or in an in vitro expression system. In some embodiments, a cell comprising a moiety has been transduced with a vector from a vector library. In some embodiments, a “genetically-perturbed cell” refers to a cell that has been transduced with a vector from a vector library.
In some embodiments, the vector comprises a moiety sequence (e.g., a second barcode). The moiety sequence was described in part (1) of this section and the embodiments disclosed therein are incorporated herein. For instance, in some embodiments, the moiety sequence is substantially complementary to the capture domain. In some embodiments, the moiety sequence is a polyadenylated sequence. In some instances, the moiety sequence of the vector comprises a sequence that uniquely identifies the vector (i.e., a UMI specific to the vector).
In some embodiments, the vector or library of vectors is a lentiviral vector. In some embodiments, a clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbation is introduced into a cell. For example, a CRISPR lentiviral vector can be used to introduce a genetic perturbation into a cell. In some embodiments, a CRISPR lentiviral vector can include a guide RNA (gRNA). In some embodiments, a CRISPR lentiviral vector can include a single guide RNA (sgRNA).
In some embodiments, pluralities of genetically-perturbed cells can be produced using a library of lentiviral vectors. In some embodiments, cells can be transduced with a library of lentiviral vectors to form genetically-perturbed cells, and the genetically-perturbed cells can be selected from the cells that did not receive a lentiviral vector (see, for example,
In some embodiments a clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbation is introduced into a cell. For example, a CRISPR lentiviral vector can be used to introduce a genetic perturbation into a cell. In some embodiments, a CRISPR lentiviral vector can include a guide RNA (gRNA). In some embodiments, a CRISPR lentiviral vector can include a single guide RNA (sgRNA).
In some embodiments, the vector in the lentiviral library includes a barcode sequence. In some embodiments, the vector includes a barcode sequence. In some embodiments, a vector includes more than one unique barcode (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, about 15, about 20, about 30, about 40, or about 50 unique barcodes). In some embodiments, a vector includes multiple copies of a unique barcode (e.g., about 10 copies, about 50, about 100, about 500, about 1000 or more). In some embodiments, identification of the barcode provides information regarding the spatial location of a particular biological analyte. In some embodiments, the barcode includes a capture domain sequence as disclosed herein. In some embodiments, the capture domain sequence is a poly(dT) sequence. In some embodiments, the capture domain sequence is a degenerate sequence. In some embodiments, the capture domain sequence is particular to a target sequence of interest. In some embodiments, the barcode includes a cleavage domain as disclosed herein. In some embodiments, the barcode includes a functional domain as disclosed herein. In some embodiments, the functional domain is a primer sequence. In some embodiments, the barcode includes a spatial barcode as disclosed herein. In some embodiments, the barcode includes a unique molecular identifier (UMI) as disclosed herein.
In some embodiments, a vector as disclosed herein expresses a gene of interest. In some embodiments, the gene of interest encodes for a protein that functions in a cellular pathway. For example, in some embodiments, the vector encodes for an inhibitor, antagonist, or agonist of various cellular pathways, including for example, pathways involving DNA damage/DNA repair, cell cycle/checkpoints, JAK/STAT signaling, MAPK signaling, GPCR/G protein, angiogenesis, immunology and inflammation, endocrinology and hormones, cancer, metabolism, and stem cells.
(c) Biological Samples
Methods disclosed herein can be performed on any type of sample. In some embodiments, the sample is a fresh tissue. In some embodiments, the sample is a frozen sample. In some embodiments, the sample was previously frozen. In some embodiments, the sample is a formalin-fixed, paraffin embedded (FFPE) sample.
Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., cancer) or a pre-disposition to a disease, and/or individuals that are in need of therapy or suspected of needing therapy. In some instances, the biological sample can include one or more diseased cells. A diseased cell can have altered metabolic properties, gene expression, protein expression, and/or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer. In some instances, the biological sample includes cancer or tumor cells. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells. In some instances, the biological sample is a heterogenous sample. In some instances, the biological sample is a heterogenous sample that includes tumor or cancer cells and/or stromal cells,
In some instances, the cancer is breast cancer. In some instances, the breast cancer is triple positive breast cancer (TPBC). In some instances, the breast cancer is triple negative breast cancer (TNBC).
In some instances, the cancer is colorectal cancer. In some instances, the cancer is ovarian cancer. In certain embodiments, the cancer is squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's or non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, myeloma, salivary gland carcinoma, kidney cancer, basal cell carcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer, testicular cancer, esophageal cancer, or a type of head or neck cancer. In certain embodiments, the cancer treated is desmoplastic melanoma, inflammatory breast cancer, thymoma, rectal cancer, anal cancer, or surgically treatable or non-surgically treatable brain stem glioma. In some embodiments, the subject is a human.
FFPE samples generally are heavily cross-linked and fragmented, and therefore this type of sample allows for limited RNA recovery using conventional detection techniques. In certain embodiments, methods of targeted RNA capture provided herein are less affected by RNA degradation associated with FFPE fixation than other methods (e.g., methods that take advantage of oligo-dT capture and reverse transcription of mRNA). In certain embodiments, methods provided herein enable sensitive measurement of specific genes of interest that otherwise might be missed with a whole transcriptomic approach.
In some instances, FFPE samples are stained (e.g., using H&E). The methods disclosed herein are compatible with H&E will allow for morphological context overlaid with transcriptomic analysis. However, depending on the need some samples may be stained with only a nuclear stain, such as staining a sample with only hematoxylin and not eosin, when location of a cell nucleus is needed.
In some embodiments, a biological sample (e.g. tissue section) can be fixed with methanol, stained with hematoxylin and eosin, and imaged. In some embodiments, fixing, staining, and imaging occurs before one or more probes are hybridized to the sample. Some embodiments of any of the workflows described herein can further include a destaining step (e.g., a hematoxylin and eosin destaining step), after imaging of the sample and prior to permeabilizing the sample. For example, destaining can be performed by performing one or more (e.g., one, two, three, four, or five) washing steps (e.g., one or more (e.g., one, two, three, four, or five) washing steps performed using a buffer including HCl). The images can be used to map spatial gene expression patterns back to the biological sample. A permeabilization enzyme can be used to permeabilize the biological sample directly on the slide.
In some embodiments, the FFPE sample is deparaffinized, permeabilized, equilibrated, and blocked before target probe oligonucleotides are added. In some embodiments, deparaffinization using xylenes. In some embodiments, deparaffinization includes multiple washes with xylenes. In some embodiments, deparaffinization includes multiple washes with xylenes followed by removal of xylenes using multiple rounds of graded alcohol followed by washing the sample with water. In some aspects, the water is deionized water. In some embodiments, equilibrating and blocking includes incubating the sample in a pre-Hyb buffer. In some embodiments, the pre-Hyb buffer includes yeast tRNA. In some embodiments, permeabilizing a sample includes washing the sample with a phosphate buffer. In some embodiments, the buffer is PBS. In some embodiments, the buffer is PBST.
For the purpose of this disclosure, an “analyte” can include any biological substance, structure, moiety, or component to be analyzed. The term “target” can similarly refer to an analyte of interest. Analytes can be broadly classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes. Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral proteins (e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, viral spike, etc.), extracellular and intracellular proteins, antibodies, and antigen binding fragments. In some embodiments, analyte(s) can be peptides or proteins, including without limitation antibodies and enzymes. In some embodiments, the analyte is a protein.
(d) Imaging and Preparation of Biological a Sample
(i) Imaging and Staining
Prior to addition of the probes, in some instances, biological samples can be stained using a wide variety of stains and staining techniques. In some instances, the biological sample is a section on a slide (e.g., a 10 μm section). In some instances, the biological sample is dried after placement onto a glass slide. In some instances, the biological sample is dried at 42° C. In some instances, drying occurs for about 1 hour, about 2, hours, about 3 hours, or until the sections become transparent. In some instances, the biological sample can be dried overnight (e.g., in a desiccator at room temperature).
In some embodiments, a sample can be stained using any number of biological stains, including but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or safranin. In some instances, the methods disclosed herein include imaging the biological sample. In some instances, imaging the sample occurs prior to deaminating the biological sample. In some instances, the sample can be stained using known staining techniques, including Can-Grunwald, Giemsa, hematoxylin and eosin (H&E), Jenner's, Leishman, Masson's trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright's, and/or Periodic Acid Schiff (PAS) staining techniques. PAS staining is typically performed after formalin or acetone fixation. In some instances, the stain is an H&E stain.
In some embodiments, the biological sample can be stained using a detectable label (e.g., radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds, and dyes) as described elsewhere herein. In some embodiments, a biological sample is stained using only one type of stain or one technique. In some embodiments, staining includes biological staining techniques such as H&E staining. In some embodiments, staining includes identifying analytes using fluorescently-conjugated antibodies. In some embodiments, a biological sample is stained using two or more different types of stains, or two or more different staining techniques. For example, a biological sample can be prepared by staining and imaging using one technique (e.g., H&E staining and brightfield imaging), followed by staining and imaging using another technique (e.g., IHC/IF staining and fluorescence microscopy) on the same biological sample.
In some embodiments, biological samples can be destained. Methods of destaining or discoloring a biological sample are known in the art, and generally depend on the nature of the stain(s) applied to the sample. For example, H&E staining can be destained by washing the sample in HCl, or any other acid (e.g., selenic acid, sulfuric acid, hydroiodic acid, benzoic acid, carbonic acid, malic acid, phosphoric acid, oxalic acid, succinic acid, salicylic acid, tartaric acid, sulfurous acid, trichloroacetic acid, hydrobromic acid, hydrochloric acid, nitric acid, orthophosphoric acid, arsenic acid, selenous acid, chromic acid, citric acid, hydrofluoric acid, nitrous acid, isocyanic acid, formic acid, hydrogen selenide, molybdic acid, lactic acid, acetic acid, carbonic acid, hydrogen sulfide, or combinations thereof). In some embodiments, destaining can include 1, 2, 3, 4, 5, or more washes in an acid (e.g., HCl). In some embodiments, destaining can include adding HCl to a downstream solution (e.g., permeabilization solution). In some embodiments, destaining can include dissolving an enzyme used in the disclosed methods (e.g., pepsin) in an acid (e.g., HCl) solution. In some embodiments, after destaining hematoxylin with an acid, other reagents can be added to the destaining solution to raise the pH for use in other applications. For example, SDS can be added to an acid destaining solution in order to raise the pH as compared to the acid destaining solution alone. As another example, in some embodiments, one or more immunofluorescence stains are applied to the sample via antibody coupling. Such stains can be removed using techniques such as cleavage of disulfide linkages via treatment with a reducing agent and detergent washing, chaotropic salt treatment, treatment with antigen retrieval solution, and treatment with an acidic glycine buffer. Methods for multiplexed staining and destaining are described, for example, in Bolognesi et al., J. Histochem. Cytochem. 2017; 65(8): 431-444, Lin et al., Nat Commun. 2015; 6:8390, Pirici et al., J. Histochem. Cytochem. 2009; 57:567-75, and Glass et al., J. Histochem. Cytochem. 2009; 57:899-905, the entire contents of each of which are incorporated herein by reference.
In some embodiments, immunofluorescence or immunohistochemistry protocols (direct and indirect staining techniques) can be performed as a part of, or in addition to, the exemplary spatial workflows presented herein. For example, tissue sections can be fixed according to methods described herein. The biological sample can be transferred to an array (e.g., capture probe array), wherein analytes (e.g., proteins) are probed using immunofluorescence protocols. For example, the sample can be rehydrated, blocked, and permeabilized (3×SSC, 2% BSA, 0.1% Triton X, 1 U/μl RNAse inhibitor for 10 minutes at 4° C.) before being stained with fluorescent primary antibodies (1:100 in 3×SSC, 2% BSA, 0.1% Triton X, 1 U/μl RNAse inhibitor for 30 minutes at 4° C.). The biological sample can be washed, coverslipped (in glycerol+1 U/μl RNAse inhibitor), imaged (e.g., using a confocal microscope or other apparatus capable of fluorescent detection), washed, and processed according to analyte capture or spatial workflows described herein.
In some instances, a glycerol solution and a cover slip can be added to the sample. In some instances, the glycerol solution can include a counterstain (e.g., DAPI).
As used herein, an antigen retrieval buffer can improve antibody capture in IF/IHC protocols. An exemplary protocol for antigen retrieval can be preheating the antigen retrieval buffer (e.g., to 95° C.), immersing the biological sample in the heated antigen retrieval buffer for a predetermined time, and then removing the biological sample from the antigen retrieval buffer and washing the biological sample.
In some embodiments, optimizing permeabilization can be useful for identifying intracellular analytes. Permeabilization optimization can include selection of permeabilization agents, concentration of permeabilization agents, and permeabilization duration. Tissue permeabilization is discussed elsewhere herein.
In some embodiments, blocking an array and/or a biological sample in preparation of labeling the biological sample decreases nonspecific binding of the antibodies to the array and/or biological sample (decreases background). Some embodiments provide for blocking buffers/blocking solutions that can be applied before and/or during application of the label, wherein the blocking buffer can include a blocking agent, and optionally a surfactant and/or a salt solution. In some embodiments, a blocking agent can be bovine serum albumin (BSA), serum, gelatin (e.g., fish gelatin), milk (e.g., non-fat dry milk), casein, polyethylene glycol (PEG), polyvinyl alcohol (PVA), or polyvinylpyrrolidone (PVP), biotin blocking reagent, a peroxidase blocking reagent, levamisole, Carnoy's solution, glycine, lysine, sodium borohydride, pontamine sky blue, Sudan Black, trypan blue, FITC blocking agent, and/or acetic acid. The blocking buffer/blocking solution can be applied to the array and/or biological sample prior to and/or during labeling (e.g., application of fluorophore-conjugated antibodies) to the biological sample.
In some embodiments, the methods described herein further include imaging the cell comprising the moiety (e.g., any of the moieties described herein). Imaging can be used, for example, to determine the morphology of the cell comprising the moiety at a distinct spatial position on the substrate. In some embodiments, the morphology is correlated to a biological analyte of the cell comprising the moiety using the methods described herein. In some embodiments, the morphology is correlated to a perturbation in the cell comprising the moiety. For example, the morphology is correlated to a change in one or more biological analytes compared to a cell including a different moiety or a cell not including the moiety.
(ii) Preparation of Samples for Application of Pooled Libraries
In some instances, the biological sample is deparaffinized. Deparaffinization can be achieved using any method known in the art. For example, in some instances, the biological sample is treated with a series of washes that include xylene and various concentrations of ethanol. In some instances, methods of deparaffinization include treatment with xylene (e.g., three washes at 5 minutes each). In some instances, the methods further include treatment with ethanol (e.g., 100% ethanol, two washes 10 minutes each; 95% ethanol, two washes 10 minutes each; 70% ethanol, two washes 10 minutes each; 50% ethanol, two washes 10 minutes each). In some instances, after ethanol washes, the biological sample can be washed with deionized water (e.g., two washes for 5 minutes each). It is appreciated that one skilled in the art can adjust these methods to optimize deparaffinization.
In some instances, the biological sample is decrosslinked. In some instances, the biological sample is decrosslinked in a solution containing TE buffer (comprising Tris and EDTA). In some instances, the TE buffer is basic (e.g., at a pH of about 9). In some instances, decrosslinking occurs at about 50° C. to about 80° C. In some instances, decrosslinking occurs at about 70° C. In some instances, decrosslinking occurs for about 1 hour at 70° C. Just prior to decrosslinking, the biological sample can be treated with an acid (e.g., 0.1M HCl for about 1 minute). After the decrosslinking step, the biological sample can be washed (e.g., with 1× PBST).
In some instances, the methods of preparing a biological sample for probe application include permeabilizing the sample. In some instances, the biological sample is permeabilized using a phosphate buffer. In some instances, the phosphate buffer is PBS (e.g., 1×PBS). In some instances, the phosphate buffer is PBST (e.g., 1×PBST). In some instances, the permeabilization step is performed multiple times (e.g., 3 times at 5 minutes each).
In some instances, the methods of preparing a biological sample for probe application include steps of equilibrating and blocking the biological sample. In some instances, equilibrating is performed using a pre-hybridization (pre-Hyb) buffer. In some instances, the pre-Hyb buffer is RNase-free. In some instances, the pre-Hyb buffer contains no bovine serum albumin (BSA), solutions like Denhardt's, or other potentially nuclease-contaminated biological materials.
In some instances, the equilibrating step is performed multiple times (e.g., 2 times at 5 minutes each; 3 times at 5 minutes each). In some instances, the biological sample is blocked with a blocking buffer. In some instances, the blocking buffer includes a carrier such as tRNA, for example yeast tRNA such as from brewer's yeast (e.g., at a final concentration of 10-20 μg/mL). In some instances, blocking can be performed for 5, 10, 15, 20, 25, or 30 minutes.
Any of the foregoing steps can be optimized for performance. For example, one can vary the temperature. In some instances, the pre-hybridization methods are performed at room temperature. In some instances, the pre-hybridization methods are performed at 4° C. (in some instances, varying the timeframes provided herein).
(e) Manipulation of Biological Samples using Pooled Libraries
(i) Manipulation of Biological Samples using a Pooled Small Molecule Library
Methods of identifying the small molecule introduced into the cell are also known to one of skill in the art. For example, in some embodiments, a moiety sequence (e.g., a second barcode) can be introduced into the cell using a particle (e.g., the particle includes the small molecule). In some embodiments, the particle further includes a moiety sequence. In some embodiments, the particle includes more than one unique moiety sequence (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, about 15, about 20, about 30, about 40, about 50, about 75, or about 100 unique moiety sequences). In some embodiments, the particle includes multiple copies of a unique moiety sequence (e.g., about 10, about 50, about 100, about 500, about 1000 or more). In some embodiments, the moiety sequence is a nucleotide sequence that identifies the small molecule. In some embodiments, identification of the moiety sequence provides information regarding the spatial location of a particular biological analyte. In some embodiments, the moiety sequence includes a capture domain sequence as disclosed herein. In some embodiments, the capture domain sequence is a poly(dT) sequence. In some embodiments, the capture domain sequence is a degenerate sequence. In some embodiments, the capture domain sequence is particular to a target sequence of interest. In some embodiment, the moiety sequence includes a cleavage domain as disclosed herein. In some embodiments, the moiety sequence includes a functional domain as disclosed herein. In some embodiments, the functional domain is a primer sequence. In some embodiment, the moiety sequence includes a spatial barcode as disclosed herein. In some embodiment, the moiety sequence includes a unique molecular identifier (UMI) as disclosed herein.
Determining the identity of the moiety sequence (e.g., second barcode) that was introduced into the cell can be used to identify the small molecule introduced into the cell. In some embodiments, the moiety sequence can be part of a polyadenylated sequence. Introducing a polyadenylated moiety sequence into the cell can allow the moiety sequence to be sequenced and identified using the methods described herein. See also, for example, Adamson et al. Cell. 167(7):1867-1882.e21, 2016; Datlinger et al. Nat Methods. 14(3):297-301, 2017; Jaitin et al. Cell. 167(7):1883-1896.e15, 2016; and Dixit et al. Cell. 167(7):1853-1866.e17, 2016, all of which are incorporated by reference herein in their entireties. As such, correlating a biological analyte from a cell including a small molecule with a first barcode of a capture probe at a distinct spatial position of a substrate can further include correlating the small molecule of the cell to a distinct spatial position of the substrate using the moiety sequence that identifies the small molecule of the cell.
In some embodiments, the methods described herein can include profiling biological analytes from a cell (or a group of cells) that includes one or more small molecules (or a library of small molecules that are all identical or that are different).
In some embodiments, the methods described herein can include one or more pluralities of cells including one or more small molecules (e.g., a library of cells including small molecules). For example, the methods described herein can be useful in detecting one or more biological analytes in cells perturbed by a library of small molecules, e.g., methods using one or more pools of small molecules. Pooling schemes are known to those in the art, see, e.g., Kainkaryam. Curr Opin Drug Discov Devel. 2009 May; 12(3): 339-350, which is incorporated herein by reference in its entirety. Accordingly, in some embodiments, a method to spatially profile one or more biological analytes present in a library of cells including a small molecule can include contacting the library of cells including a small molecule with a substrate including a plurality of capture probes, wherein a capture probe of the plurality includes a first barcode and a capture domain; releasing the one or more biological analytes from members of the library of cells including a small molecule, wherein the one or more biological analytes are bound by capture probes at distinct spatial positions of the substrate; detecting the one or more biological analytes bound by capture probes; and correlating each biological analyte with the first barcode from the capture probe it was bound to at the distinct spatial position of the substrate; thus profiling the one or more biological analytes as present in the library of cells including a small molecule at one or more distinct spatial positions. In some embodiments, one biological analyte is bound to one capture probe.
In some embodiments, the methods described herein can include profiling biological analytes from a cell that includes multiple, identical small molecules. In some embodiments, the methods described herein can include profiling biological analytes from a cell that includes multiple, unique (i.e., different) small molecules. In some embodiments, the methods described herein can include profiling biological analytes from a group of cells that each include a single small molecule. In some embodiments, the methods described herein can include profiling biological analytes from a group of cells, each of which includes multiple copies of the same small molecule. In some embodiments, the methods described herein can include profiling biological analytes from a group of cells that each include multiple copies of different small molecules.
In some embodiments, the methods disclosed herein also include a wash step. The wash step removes any unbound probes. Wash steps could be performed between any of the steps in the methods disclosed herein. For example, a wash step can be performed after adding probes to the biological sample. As such, free/unbound probes are washed away, leaving only probes that have hybridized to an analyte. In some instances, multiple (i.e., at least 2, 3, 4, 5, or more) wash steps occur between the methods disclosed herein. Wash steps can be performed at times (e.g., 1, 2, 3, 4, or 5 minutes) and temperatures (e.g., room temperature) known in the art and determined by a person of skill in the art.
In some instances, wash steps are performed using a wash buffer. In some instances, the wash buffer includes SSC (e.g., 1×SSC). In some instances, the wash buffer includes PBS (e.g., 1×PBS). In some instances, the wash buffer includes PBST (e.g., 1×PBST). In some instances, the wash buffer can also include formamide or be formamide free.
In some embodiments, a biological sample can optionally be separated into single cells, cell groups, or other fragments/pieces that are smaller than the original, unfragmented sample. Each of these smaller portions of the sample can be analyzed to obtain spatially-resolved analyte information for the sample.
For samples that have been separated into smaller fragments—and particularly, for samples that have been disaggregated, dissociated, or otherwise separated into individual cells—one method for analyzing the fragments involves separating the fragments into individual partitions (e.g., fluid droplets), and then analyzing the contents of the partitions. In general, each partition maintains separation of its own contents from the contents of other partitions. The partition can be a droplet in an emulsion, for example.
Microfluidic channel networks (e.g., on a chip) can be utilized to generate partitions. Alternative mechanisms can also be employed in the partitioning of individual biological particles, including porous membranes through which aqueous mixtures of cells are extruded into non-aqueous fluids. In some embodiments, a microfluidical channel structure can be used for partitioning individual analytes (e.g., cells) into discrete partitions. For example, a first aqueous fluid that includes suspended biological particles (or cells) may be transported along a channel segment into a junction, while a second fluid that is immiscible with the first aqueous fluid is delivered to the junction from each of the channel segments to create discrete droplets of the first aqueous fluid flowing into a channel segment, and flowing away from the junction. The channel segment may be fluidically coupled to an outlet reservoir where the discrete droplets can be stored and/or harvested. A discrete droplet generated may include an individual biological particle. A discrete droplet generated may include more than one individual biological particle. A discrete droplet may contain no biological particle. Each discrete partition may maintain separation of its own contents (e.g., individual biological particle) from the contents of other partitions.
In some embodiments, one or more barcodes (e.g., spatial barcodes, UMIs, or a combination thereof) can be introduced into a partition as part of the analyte. As described previously, barcodes can be bound to the analyte directly, or can form part of a capture probe or analyte capture agent that is hybridized to, conjugated to, or otherwise associated with an analyte, such that when the analyte is introduced into the partition, the barcode(s) are introduced as well.
A second fluid 910 that is immiscible with the aqueous fluid 907 (e.g., oil) can be delivered to the junction 906 from each of channel segments 903 and 904. Upon meeting of the aqueous fluid 907 from each of channel segments 901 and 902 and the second fluid 910 from each of channel segments 903 and 904 at the channel junction 906, the aqueous fluid 907 can be partitioned as discrete droplets 911 in the second fluid 910 and flow away from the junction 906 along channel segment 905. The channel segment 905 can deliver the discrete droplets to an outlet reservoir fluidly coupled to the channel segment 905, where they can be harvested.
As an alternative, the channel segments 901 and 902 can meet at another junction upstream of the junction 906. At such junction, beads and biological particles can form a mixture that is directed along another channel to the junction 906 to yield droplets 911. The mixture can provide the beads and biological particles in an alternating fashion, such that, for example, a droplet includes a single bead and a single biological particle.
The second fluid 910 can include an oil, such as a fluorinated oil, that includes a fluorosurfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets 911.
The partitions described herein can include small volumes, for example, less than about 10 microliters (μL), 5 μL, 1 μL, 900 picoliters (pL), 800 pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 20 pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less. In the foregoing discussion, droplets with beads were formed at the junction of different fluid streams. In some embodiments, droplets can be formed by gravity-based partitioning methods.
An aqueous fluid 960 comprising a plurality of particles 956 may be transported along the channel segment 952 into the junction 958 to meet a second fluid 962 (e.g., oil, etc.) that is immiscible with the aqueous fluid 960 in the reservoir 954 to create droplets 964 of the aqueous fluid 960 flowing into the reservoir 954. At the junction 958 where the aqueous fluid 960 and the second fluid 962 meet, droplets can form based on factors such as the hydrodynamic forces at the junction 958, relative flow rates of the two fluids 960, 962, fluid properties, and certain geometric parameters (e.g., Δh, etc.) of the channel structure 950. A plurality of droplets can be collected in the reservoir 954 by continuously injecting the aqueous fluid 960 from the channel segment 952 at the junction 958.
A discrete droplet generated may comprise one or more particles of the plurality of particles 956. As described elsewhere herein, a particle may be any particle, such as a bead, cell bead, gel bead, biological particle, macromolecular constituents of biological particle, or other particles. Alternatively, a discrete droplet generated may not include any particles.
In some instances, the aqueous fluid 960 can have a substantially uniform concentration or frequency of particles 956. As described elsewhere herein, the particles 956 (e.g., beads) can be introduced into the channel segment 952 from a separate channel (not shown in
In some instances, the second fluid 962 may not be subjected to and/or directed to any flow in or out of the reservoir 954. For example, the second fluid 962 may be substantially stationary in the reservoir 954. In some instances, the second fluid 962 may be subjected to flow within the reservoir 954, but not in or out of the reservoir 954, such as via application of pressure to the reservoir 954 and/or as affected by the incoming flow of the aqueous fluid 960 at the junction 958. Alternatively, the second fluid 962 may be subjected and/or directed to flow in or out of the reservoir 954. For example, the reservoir 954 can be a channel directing the second fluid 962 from upstream to downstream, transporting the generated droplets.
The channel structure 950 at or near the junction 958 may have certain geometric features that at least partly determine the volumes and/or shapes of the droplets formed by the channel structure 950. The channel segment 952 can have a first cross-section height, h1, and the reservoir 954 can have a second cross-section height, h2. The first cross-section height, h1, and the second cross-section height, h2, may be different, such that at the junction 958, there is a height difference of Δh. The second cross-section height, h2, may be greater than the first cross-section height, h1. In some instances, the reservoir may thereafter gradually increase in cross-section height, for example, the more distant it is from the junction 958. In some instances, the cross-section height of the reservoir may increase in accordance with expansion angle, β, at or near the junction 958. The height difference, Δh, and/or expansion angle, β, can allow the tongue (portion of the aqueous fluid 960 leaving channel segment 952 at junction 958 and entering the reservoir 954 before droplet formation) to increase in depth and facilitate decrease in curvature of the intermediately formed droplet. For example, droplet volume may decrease with increasing height difference and/or increasing expansion angle.
The height difference, Δh, can be at least about 1 μm. Alternatively, the height difference can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 μm or more. Alternatively, the height difference can be at most about 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 μm or less. In some instances, the expansion angle, β, may be between a range of from about 0.5° to about 4°, from about 0.1° to about 10°, or from about 0° to about 90°. For example, the expansion angle can be at least about 0.01°, 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 85°, or higher. In some instances, the expansion angle can be at most about 89°, 88°, 87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.
In some instances, the flow rate of the aqueous fluid 960 entering the junction 958 can be between about 0.04 microliters (μL)/minute (min) and about 40 μL/min. In some instances, the flow rate of the aqueous fluid 960 entering the junction 958 can be between about 0.01 microliters (μL)/minute (min) and about 100 μL/min. Alternatively, the flow rate of the aqueous fluid 960 entering the junction 958 can be less than about 0.01 μL/min. alternatively, the flow rate of the aqueous fluid 960 entering the junction 958 can be greater than about 40 μL/min, such as 45 μL/min, 50 μL/min, 55 μL/min, 60 μL/min, 65 μL/min, 70 μL/min, 75 μL/min, 80 μL/min, 85 μL/min, 90 μL/min, 95 μL/min, 100 μL/min, 110 μL/min, 120 μL/min, 130 μL/min, 140 μL/min, 150 μL/min, or greater. At lower flow rates, such as flow rates of about less than or equal to 10 microliters/minute, the droplet radius may not be dependent on the flow rate of the aqueous fluid 960 entering the junction 958. The second fluid 962 may be stationary, or substantially stationary, in the reservoir 954. Alternatively, the second fluid 962 may be flowing, such as at the above flow rates described for the aqueous fluid 960.
While
It should be noted that while the example workflow in
By way of example, in the context of analyzing sample RNA as shown in
(ii) Manipulation of Biological Samples Using a Pooled Nucleic Acid Library
Methods of identifying a genetic perturbation or genetic moiety introduced into a cell are also known to one of skill in the art. See, for example, Adamson et al. Cell. 167(7):1867-1882.e21, 2016; Datlinger et al. Nat Methods. 14(3):297-301, 2017; Jaitin et al. Cell. 167(7):1883-1896.e15, 2016; and Dixit et al. Cell. 167(7):1853-1866.e17, 2016, each of which is incorporated by reference herein in its entirety.
In some instances, the methods include introducing the pooled nucleic acid library into a cell or plurality of cells. The term “introducing,” as used herein, includes delivery of a vector or pooled library to a cell or cells. Such introducing may take place in vivo, in vitro, or ex vivo. A vector for expression of a gene product may be introduced into a cell by transfection, which typically means insertion of heterologous DNA into a cell by physical means (e.g., calcium phosphate transfection, electroporation, microinjection, or lipofection); infection, which typically refers to introduction by way of an infectious agent, i.e. a virus; or transduction, which typically means stable infection of a cell with a virus or the transfer of genetic material from one microorganism to another by way of a viral agent (e.g., a bacteriophage).
After introduction, the methods in some instances can include determining the presence of a genetic moiety (i.e., a nucleic acid (e.g., gRNA and/or sgRNA)) that was introduced into a cell. For example, a moiety sequence (e.g., a second barcode) can be introduced into the genetically-perturbed cell that identifies the genetic moiety in the cell (e.g., identifies the gRNA introduced into the cell) and/or a polyadenylated nucleotide sequence, such as a polyadenylated gRNA sequence, can be introduced into the genetically-perturbed cell. In some embodiments, the moiety sequence can be part of a polyadenylated sequence. Introducing a polyadenylated barcode sequence and/or a polyadenylated gRNA sequence into the cell can allow the second barcode and/or gRNA sequence to be sequenced and identified using the methods described herein. In some embodiments, the CRISPR lentiviral vector can further include the moiety sequence identifying the gRNA and/or a polyadenylated or an oligo (dT) nucleotide sequence (e.g., a polyadenylated gRNA sequence). As such, correlating a biological analyte from a genetically-perturbed cell with a first barcode of a capture probe at a distinct spatial position of a substrate can further include correlating the genetic perturbation of the cell to a distinct spatial position of the substrate using the moiety sequence that identifies the genetic perturbation of the cell and/or the polyadenylated nucleotide sequence. In addition, also disclosed herein are methods of selecting a cell that includes the genetic perturbation.
In some embodiments, the methods described herein can include profiling biological analytes from one or more pluralities of genetically-perturbed cells (e.g., a library of genetically-perturbed cells). For example, a method to spatially profile one or more biological analytes present in a library of genetically-perturbed cells can include contacting the library of genetically-perturbed cells with a substrate including a plurality of capture probes, wherein a capture probe of the plurality includes a first barcode and a capture domain; releasing the one or more biological analytes from members of the library of genetically-perturbed cells, wherein the one or more biological analytes are bound by capture probes at distinct spatial positions of the substrate; detecting the one or more biological analytes bound by capture probes; and correlating each biological analyte with the first barcode from the capture probe it was bound to at the distinct spatial position of the substrate; thus profiling the one or more biological analytes as present in the library of genetically-perturbed cells at distinct spatial positions. In some embodiments, one biological analyte is bound to one capture probe.
In some embodiments, a plurality of genetically-perturbed cells includes a different genetic perturbation from another plurality of genetically-perturbed cells. In some embodiments, each plurality of genetically-perturbed cells includes a different genetic perturbation from each other plurality of genetically-perturbed cells. In some embodiments, a plurality of genetically-perturbed cells with a different genetic perturbation from another plurality of genetically-perturbed cells also has a different polyadenylated barcode sequence and/or a polyadenylated gRNA sequence from the other plurality of genetically-perturbed cells (e.g., the polyadenylated barcode sequence and/or a polyadenylated gRNA sequence can identify the genetic perturbation of the cell). In some embodiments, each plurality of genetically-perturbed cells that has a different genetic perturbation from each other plurality of genetically-perturbed cells also has a different polyadenylated barcode sequence and/or a polyadenylated gRNA sequence from each other plurality of genetically-perturbed cells (e.g., the polyadenylated barcode sequence and/or a polyadenylated gRNA sequence can identify the genetic perturbation of the cell).
In some embodiments, the methods disclosed herein also include a wash step. The wash step removes any unbound probes. Wash steps could be performed between any of the steps in the methods disclosed herein. For example, a wash step can be performed after adding probes to the biological sample. As such, free/unbound probes are washed away, leaving only probes that have hybridized to an analyte. In some instances, multiple (i.e., at least 2, 3, 4, 5, or more) wash steps occur between the methods disclosed herein. Wash steps can be performed at times (e.g., 1, 2, 3, 4, or 5 minutes) and temperatures (e.g., room temperature) known in the art and determined by a person of skill in the art.
In some instances, wash steps are performed using a wash buffer. In some instances, the wash buffer includes SSC (e.g., 1×SSC). In some instances, the wash buffer includes PBS (e.g., 1×PBS). In some instances, the wash buffer includes PBST (e.g., 1×PBST). In some instances, the wash buffer can also include formamide or be formamide free.
In some embodiments, a biological sample can optionally be separated into single cells, cell groups, or other fragments/pieces that are smaller than the original, unfragmented sample. Each of these smaller portions of the sample can be analyzed to obtain spatially-resolved analyte information for the sample.
For samples that have been separated into smaller fragments—and particularly, for samples that have been disaggregated, dissociated, or otherwise separated into individual cells—one method for analyzing the fragments involves separating the fragments into individual partitions (e.g., fluid droplets), and then analyzing the contents of the partitions. In general, each partition maintains separation of its own contents from the contents of other partitions. The partition can be a droplet in an emulsion, for example.
Microfluidic channel networks (e.g., on a chip) can be utilized to generate partitions. Alternative mechanisms can also be employed in the partitioning of individual biological particles, including porous membranes through which aqueous mixtures of cells are extruded into non-aqueous fluids. In some embodiments, a microfluidical channel structure can be used for partitioning individual analytes (e.g., cells) into discrete partitions. For example, a first aqueous fluid that includes suspended biological particles (or cells) may be transported along a channel segment into a junction, while a second fluid that is immiscible with the first aqueous fluid is delivered to the junction from each of the channel segments to create discrete droplets of the first aqueous fluid flowing into a channel segment, and flowing away from the junction. The channel segment may be fluidically coupled to an outlet reservoir where the discrete droplets can be stored and/or harvested. A discrete droplet generated may include an individual biological particle. A discrete droplet generated may include more than one individual biological particle. A discrete droplet may contain no biological particle. Each discrete partition may maintain separation of its own contents (e.g., individual biological particle) from the contents of other partitions. Microfluidic systems have been described in part (1) of this section and is incorporated herein.
(e) Spatial Detection of Manipulated Cells
After an analyte and/or moiety from the cell, e.g., a cell in a biological sample, has hybridized or otherwise been associated with a capture probe according to any of the methods described above in connection with the general spatial cell-based analytical methodology, the barcoded constructs that result from hybridization/association are analyzed.
In some embodiments, after contacting a biological sample with a substrate that includes capture probes, a removal step can optionally be performed to remove all or a portion of the biological sample from the substrate. In some embodiments, the removal step includes enzymatic and/or chemical degradation of cells of the biological sample. For example, the removal step can include treating the biological sample with an enzyme (e.g., a proteinase, e.g., proteinase K) to remove at least a portion of the biological sample from the substrate. In some embodiments, the removal step can include ablation of the tissue (e.g., laser ablation).
In some embodiments, provided herein are methods for spatially detecting an analyte (e.g., detecting the location of an analyte, e.g., a biological analyte) from a biological sample (e.g., present in a biological sample), the method comprising: (a) optionally staining and/or imaging a biological sample on a substrate; (b) permeabilizing (e.g., providing a solution comprising a permeabilization reagent to) the biological sample on the substrate; (c) contacting the biological sample with an array comprising a plurality of capture probes, wherein a capture probe of the plurality captures the biological analyte; and (d) analyzing the captured biological analyte, thereby spatially detecting the biological analyte; wherein the biological sample is fully or partially removed from the substrate.
In some embodiments, a biological sample is not removed from the substrate. For example, the biological sample is not removed from the substrate prior to releasing a capture probe (e.g., a capture probe bound to an analyte) from the substrate. In some embodiments, such releasing comprises cleavage of the capture probe from the substrate (e.g., via a cleavage domain). In some embodiments, such releasing does not comprise releasing the capture probe from the substrate (e.g., a copy of the capture probe bound to an analyte can be made and the copy can be released from the substrate, e.g., via denaturation). In some embodiments, the biological sample is not removed from the substrate prior to analysis of an analyte bound to a capture probe after it is released from the substrate. In some embodiments, the biological sample remains on the substrate during removal of a capture probe from the substrate and/or analysis of an analyte bound to the capture probe after it is released from the substrate. In some embodiments, the biological sample remains on the substrate during removal (e.g., via denaturation) of a copy of the capture probe (e.g., complement). In some embodiments, analysis of an analyte bound to a capture probe from the substrate can be performed without subjecting the biological sample to enzymatic and/or chemical degradation of the cells (e.g., permeabilized cells) or ablation of the tissue (e.g., laser ablation).
In some embodiments, at least a portion of the biological sample is not removed from the substrate. For example, a portion of the biological sample can remain on the substrate prior to releasing a capture probe (e.g., a capture prove bound to an analyte) from the substrate and/or analyzing an analyte bound to a capture probe released from the substrate. In some embodiments, at least a portion of the biological sample is not subjected to enzymatic and/or chemical degradation of the cells (e.g., permeabilized cells) or ablation of the tissue (e.g., laser ablation) prior to analysis of an analyte bound to a capture probe from the substrate.
In some embodiments, provided herein are methods for spatially detecting an analyte (e.g., detecting the location of an analyte, e.g., a biological analyte) from a biological sample (e.g., present in a biological sample) that include: (a) optionally staining and/or imaging a biological sample on a substrate; (b) permeabilizing (e.g., providing a solution comprising a permeabilization reagent to) the biological sample on the substrate; (c) contacting the biological sample with an array comprising a plurality of capture probes, wherein a capture probe of the plurality captures the biological analyte; and (d) analyzing the captured biological analyte, thereby spatially detecting the biological analyte; where the biological sample is not removed from the substrate.
In some embodiments, provided herein are methods for spatially detecting a biological analyte of interest from a biological sample that include: (a) staining and imaging a biological sample on a substrate; (b) providing a solution comprising a permeabilization reagent to the biological sample on the substrate; (c) contacting the biological sample with an array on a substrate, wherein the array comprises one or more capture probe pluralities thereby allowing the one or more pluralities of capture probes to capture the biological analyte of interest; and (d) analyzing the captured biological analyte, thereby spatially detecting the biological analyte of interest; where the biological sample is not removed from the substrate.
In some embodiments, the method further includes subjecting a region of interest in the biological sample to spatial transcriptomic analysis. In some embodiments, one or more of the capture probes includes a capture domain. In some embodiments, one or more of the capture probes comprises a unique molecular identifier (UMI). In some embodiments, one or more of the capture probes comprises a cleavage domain. In some embodiments, the cleavage domain comprises a sequence recognized and cleaved by uracil-DNA glycosylase, apurinic/apyrimidinic (AP) endonuclease (APE1), uracil-specific excision reagent (USER), and/or an endonuclease VIII. In some embodiments, one or more capture probes do not comprise a cleavage domain and is not cleaved from the array.
In some embodiments, a capture probe can be extended (an “extended capture probe,” e.g., as described herein). For example, extending a capture probe can include generating cDNA from a captured (hybridized) RNA. This process involves synthesis of a complementary strand of the hybridized nucleic acid, e.g., generating cDNA based on the captured RNA template (the RNA hybridized to the capture domain of the capture probe). Thus, in an initial step of extending a capture probe, e.g., the cDNA generation, the captured (hybridized) nucleic acid, e.g., RNA, acts as a template for the extension, e.g., reverse transcription, step.
In some embodiments, the capture probe is extended using reverse transcription. For example, reverse transcription includes synthesizing cDNA (complementary or copy DNA) from RNA, e.g., (messenger RNA), using a reverse transcriptase. In some embodiments, reverse transcription is performed while the tissue is still in place, generating an analyte library, where the analyte library includes the spatial barcodes from the adjacent capture probes. In some embodiments, the capture probe is extended using one or more DNA polymerases.
In some embodiments, a capture domain of a capture probe includes a primer for producing the complementary strand of a nucleic acid hybridized to the capture probe, e.g., a primer for DNA polymerase and/or reverse transcription. The nucleic acid, e.g., DNA and/or cDNA, molecules generated by the extension reaction incorporate the sequence of the capture probe. The extension of the capture probe, e.g., a DNA polymerase and/or reverse transcription reaction, can be performed using a variety of suitable enzymes and protocols.
In some embodiments, a full-length DNA (e.g., cDNA) molecule is generated. In some embodiments, a “full-length” DNA molecule refers to the whole of the captured nucleic acid molecule. However, if a nucleic acid (e.g., RNA) was partially degraded in the tissue sample, then the captured nucleic acid molecules will not be the same length as the initial RNA in the tissue sample. In some embodiments, the 3′ end of the extended probes, e.g., first strand cDNA molecules, is modified. For example, a linker or adaptor can be ligated to the 3′ end of the extended probes. This can be achieved using single stranded ligation enzymes such as T4 RNA ligase or Circligase™ (available from Lucigen, Middleton, Wis.). In some embodiments, template switching oligonucleotides are used to extend cDNA in order to generate a full-length cDNA (or as close to a full-length cDNA as possible). In some embodiments, a second strand synthesis helper probe (a partially double stranded DNA molecule capable of hybridizing to the 3′ end of the extended capture probe), can be ligated to the 3′ end of the extended probe, e.g., first strand cDNA, molecule using a double stranded ligation enzyme such as T4 DNA ligase. Other enzymes appropriate for the ligation step are known in the art and include, e.g., Tth DNA ligase, Taq DNA ligase, Thermococcus sp. (strain 9° N) DNA ligase (9° N™ DNA ligase, New England Biolabs), Ampligase™ (available from Lucigen, Middleton, Wis.), and SplintR (available from New England Biolabs, Ipswich, Mass.). In some embodiments, a polynucleotide tail, e.g., a poly(A) tail, is incorporated at the 3′ end of the extended probe molecules. In some embodiments, the polynucleotide tail is incorporated using a terminal transferase active enzyme.
In some embodiments, double-stranded extended capture probes are treated to remove any unextended capture probes prior to amplification and/or analysis, e.g., sequence analysis. This can be achieved by a variety of methods, e.g., using an enzyme to degrade the unextended probes, such as an exonuclease enzyme, or purification columns.
In some embodiments, extended capture probes are amplified to yield quantities that are sufficient for analysis, e.g., via DNA sequencing. In some embodiments, the first strand of the extended capture probes (e.g., DNA and/or cDNA molecules) acts as a template for the amplification reaction (e.g., a polymerase chain reaction).
In some embodiments, the amplification reaction incorporates an affinity group onto the extended capture probe (e.g., RNA-cDNA hybrid) using a primer including the affinity group. In some embodiments, the primer includes an affinity group and the extended capture probes includes the affinity group. The affinity group can correspond to any of the affinity groups described previously.
In some embodiments, the extended capture probes including the affinity group can be coupled to a substrate specific for the affinity group. In some embodiments, the substrate can include an antibody or antibody fragment. In some embodiments, the substrate includes avidin or streptavidin and the affinity group includes biotin. In some embodiments, the substrate includes maltose and the affinity group includes maltose-binding protein. In some embodiments, the substrate includes maltose-binding protein and the affinity group includes maltose. In some embodiments, amplifying the extended capture probes can function to release the extended probes from the surface of the substrate, insofar as copies of the extended probes are not immobilized on the substrate.
In some embodiments, the extended capture probe or complement or amplicon thereof is released. The step of releasing the extended capture probe or complement or amplicon thereof from the surface of the substrate can be achieved in a number of ways. In some embodiments, an extended capture probe or a complement thereof is released from the array by nucleic acid cleavage and/or by denaturation (e.g., by heating to denature a double-stranded molecule).
In some embodiments, the extended capture probe or complement or amplicon thereof is released from the surface of the substrate (e.g., array) by physical means. For example, where the extended capture probe is indirectly immobilized on the array substrate, e.g., via hybridization to a surface probe, it can be sufficient to disrupt the interaction between the extended capture probe and the surface probe. Methods for disrupting the interaction between nucleic acid molecules include denaturing double stranded nucleic acid molecules are known in the art. A straightforward method for releasing the DNA molecules (i.e., of stripping the array of extended probes) is to use a solution that interferes with the hydrogen bonds of the double stranded molecules. In some embodiments, the extended capture probe is released by an applying heated solution, such as water or buffer, of at least 85° C., e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99° C. In some embodiments, a solution including salts, surfactants, etc. that can further destabilize the interaction between the nucleic acid molecules is added to release the extended capture probe from the substrate.
In some embodiments, where the extended capture probe includes a cleavage domain, the extended capture probe is released from the surface of the substrate by cleavage. For example, the cleavage domain of the extended capture probe can be cleaved by any of the methods described herein. In some embodiments, the extended capture probe is released from the surface of the substrate, e.g., via cleavage of a cleavage domain in the extended capture probe, prior to the step of amplifying the extended capture probe.
In some embodiments, probes complementary to the extended capture probe can be contacted with the substrate. In some embodiments, the biological sample can be in contact with the substrate when the probes are contacted with the substrate. In some embodiments, the biological sample can be removed from the substrate prior to contacting the substrate with probes. In some embodiments, the probes can be labeled with a detectable label (e.g., any of the detectable labels described herein). In some embodiments, probes that do not specially bind (e.g., hybridize) to an extended capture probe can be washed away. In some embodiments, probes complementary to the extended capture probe can be detected on the substrate (e.g., imaging, any of the detection methods described herein).
In some embodiments, probes complementary to an extended capture probe can be about 4 nucleotides to about 100 nucleotides long. In some embodiments, probes (e.g., detectable probes) complementary to an extended capture probe can be about 10 nucleotides to about 90 nucleotides long. In some embodiments, probes (e.g., detectable probes) complementary to an extended capture probe can be about 20 nucleotides to about 80 nucleotides long. In some embodiments, probes (e.g., detectable probes) complementary to an extended capture probe can be about 30 nucleotides to about 60 nucleotides long. In some embodiments, probes (e.g., detectable probes) complementary to an extended capture probe can be about 40 nucleotides to about 50 nucleotides long. In some embodiments, probes (e.g., detectable probes) complementary to an extended capture probe can be about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, and about 99 nucleotides long.
In some embodiments, about 1 to about 100 probes can be contacted to the substrate and specifically bind (e.g., hybridize) to an extended capture probe. In some embodiments, about 1 to about 10 probes can be contacted to the substrate and specifically bind (e.g., hybridize) to an extended capture probe. In some embodiments, about 10 to about 100 probes can be contacted to the substrate and specifically bind (e.g., hybridize) to an extended capture probe. In some embodiments, about 20 to about 90 probes can be contacted to the substrate and specifically bind (e.g., hybridize) to an extended capture probe. In some embodiments, about 30 to about 80 probes (e.g., detectable probes) can be contacted to the substrate and specifically bind (e.g., hybridize) to an extended capture probe. In some embodiments, about 40 to about 70 probes can be contacted to the substrate and specifically bind (e.g., hybridize) to an extended capture probe. In some embodiments, about 50 to about 60 probes can be contacted to the substrate and specifically bind (e.g., hybridize) to an extended capture probe. In some embodiments, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, and about 99 probes can be contacted to the substrate and specifically bind (e.g., hybridize) to an extended capture probe.
In some embodiments, the probes can be complementary to a single analyte (e.g., a single gene). In some embodiments, the probes can be complementary to one or more analytes (e.g., analytes in a family of genes). In some embodiments, the probes (e.g., detectable probes) can be for a panel of genes associated with a disease (e.g., cancer, Alzheimer's disease, Parkinson's disease).
In some instances, the ligated probe and capture probe can be amplified or copied, creating a plurality of cDNA molecules. In some embodiments, cDNA can be denatured from the capture probe template and transferred (e.g., to a clean tube) for amplification, and/or library construction. The spatially-barcoded cDNA can be amplified via PCR prior to library construction. The cDNA can then be enzymatically fragmented and size-selected in order to optimize for cDNA amplicon size. P5 and P7 sequences directed to capturing the amplicons on a sequencing flowcell (e.g., Illumina sequencing instruments) can be appended to the amplicons, i7, and i5 can be used as sample indexes, and TruSeq Read 2 can be added via End Repair, A-tailing, Adaptor Ligation, and PCR. The cDNA fragments can then be sequenced using paired-end sequencing using TruSeq Read 1 and TruSeq Read 2 as sequencing primer sites. A skilled artisan will understand that additional or alternative sequences used by other sequencing instruments or technologies are also equally applicable for use in the aforementioned methods as the current methods are not limited to any a particular sequencing platform.
In some embodiments, where a sample is barcoded directly via hybridization with capture probes or analyte capture agents hybridized, bound, or associated with either the cell surface, or introduced into the cell, as described above, sequencing can be performed on the intact sample.
A wide variety of different sequencing methods can be used to analyze the barcoded analyte or moiety. In general, sequenced polynucleotides can be, for example, nucleic acid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (e.g., single stranded DNA or DNA/RNA hybrids, and nucleic acid molecules with a nucleotide analog).
Sequencing of polynucleotides can be performed by various systems. More generally, sequencing can be performed using nucleic acid amplification, polymerase chain reaction (PCR) (e.g., digital PCR and droplet digital PCR (ddPCR), quantitative PCR, real time PCR, multiplex PCR, PCR-based single plex methods, emulsion PCR), and/or isothermal amplification. Non-limiting examples of methods for sequencing genetic material include, but are not limited to, DNA hybridization methods (e.g., Southern blotting), restriction enzyme digestion methods, Sanger sequencing methods, next-generation sequencing methods (e.g., single-molecule real-time sequencing, nanopore sequencing, and Polony sequencing), ligation methods, and microarray methods.
In some embodiments, a capture probe capture domain is blocked prior to adding a second probe oligonucleotide to a cell, e.g., a cell in a biological sample. This prevents the capture probe capture domain from prematurely hybridizing to the capture domain.
In some embodiments, a blocking probe is used to block or modify the free 3′ end of the capture probe capture domain. In some embodiments, a blocking probe can be hybridized to the capture probe capture domain of the second probe to mask the free 3′ end of the capture probe capture domain. In some embodiments, a blocking probe can be a hairpin probe or partially double stranded probe. In some embodiments, the free 3′ end of the capture probe capture domain of the second probe can be blocked by chemical modification, e.g., addition of an azidomethyl group as a chemically reversible capping moiety such that the capture probes do not include a free 3′ end. Blocking or modifying the capture probe capture domain, particularly at the free 3′ end of the capture probe capture domain, prior to contacting second probe with the substrate, prevents hybridization of the second probe to the capture domain (e.g., prevents the capture of a poly(A) of a capture probe capture domain to a poly(T) capture domain). In some embodiments, a blocking probe can be referred to as a capture probe capture domain blocking moiety.
In some embodiments, the blocking probes can be reversibly removed. For example, blocking probes can be applied to block the free 3′ end of either or both the capture probe capture domain and/or the capture probes. Blocking interaction between the capture probe capture domain and the capture probe on the substrate can reduce non-specific capture to the capture probes. After the second probe hybridizes to the analyte and is ligated to a first probe, one or more spanning probes, or a third oligonucleotide, the blocking probes can be removed from the 3′ end of the capture probe capture domain and/or the capture probe, and the ligation product can migrate to and become bound by the capture probes on the substrate. In some embodiments, the removal includes denaturing the blocking probe from capture probe capture domain and/or capture probe. In some embodiments, the removal includes removing a chemically reversible capping moiety. In some embodiments, the removal includes digesting the blocking probe with an RNase (e.g., RNase H).
In some embodiments, the blocking probes are oligo (dT) blocking probes. In some embodiments, the oligo (dT) blocking probes can have a length of 15-30 nucleotides. In some embodiments, the oligo (dT) blocking probes can have a length of 10-50 nucleotides, e.g., 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-50, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 20-50, 20-45, 20-40, 20-35, 20-30, 20-25, 25-50, 25-45, 25-40, 25-35, 25-30, 30-50, 30-45, 30-40, 30-35, 35-50, 35-45, 35-40, 40-50, 40-45, or 45-50 nucleotides. In some embodiments, the analyte capture agents can be blocked at different temperatures (e.g., 4° C. and 37° C.).
(f) Kits and Compositions
In some embodiments, also provided herein are kits and compositions that include one or more reagents to detect one or more analytes and/or one or more moieties described herein. In some instances, the kit includes a substrate comprising a plurality of capture probes comprising a spatial barcode and the capture domain. In some instances, the kit includes a plurality of probes (e.g., a first probe, a second probe, one or more spanning probes, and/or a third oligonucleotide).
A non-limiting example of a kit used to perform any of the methods described herein includes: (a) a substrate comprising a plurality of capture probes comprising a spatial barcode and a capture domain; (b) a system comprising: a plurality of first probes and second probes, wherein a first probe and a second probe each comprises sequences that are substantially complementary to an analyte, and wherein the second probe comprises a capture binding domain; and (c) instructions for performing the method of any one of the preceding claims.
Another non-limiting example of a kit used to perform any of the methods described herein includes: (a) an array comprising a plurality of capture probes; (b) a plurality of probes comprising a first probe and a second, wherein the first probe and the second probe are substantially complementary to adjacent sequences of an analyte, wherein the second probe comprises (i) a capture probe binding domain that is capable of binding to a capture domain of the capture probe and (ii) a linker sequence; (c) a plurality of enzymes comprising a ribonuclease and a ligase; and (d) instructions for performing the method of any one of the preceding claims.
Another non-limiting example of a kit used to perform any of the methods described herein includes: (a) an array comprising a plurality of capture probes; (b) a plurality of probes comprising a first probe and a second probe, wherein the first probe and the second probe are substantially complementary to adjacent sequences of an analyte, wherein the first probe includes a linker sequence, wherein the second probe comprises a capture probe binding domain that is capable of binding to a capture domain of the capture probe; (c) a plurality of enzymes comprising a ribonuclease and a ligase; and (d) instructions for performing the method of any one of the preceding claims.
A library of small molecules with particular targets is selected. The small molecules are loaded into a 96-well plate, and particles that encapsulate the small molecules are added to each well. Particles are barcoded with a nucleic acid barcode (e.g., a moiety sequence) such that there is a predetermined 1:1 mapping between the small molecule and the barcode sequence (e.g., moiety sequence) present in each particle. After barcoding, a nucleic acid barcoded small molecule library is generated. See
A small molecule library of Example 1 is incubated with a cell line, allowing the small molecule to penetrate the cell. See
A small molecule library of Example 1 is incubated with a cell line, allowing the small molecule to penetrate the cell. See
A lentiviral library is selected. The lentiviral library is transduced into cells, generating a genetically-perturbed cells. See
This application claims priority to U.S. Provisional Patent Application No. 62/963,897, filed Jan. 21, 2020; and U.S. Provisional Patent Application No. 62/963,879, filed Jan. 21, 2020. The contents of each of these applications are incorporated herein by reference in their entireties.
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| Number | Date | Country | |
|---|---|---|---|
| 20210222242 A1 | Jul 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62963879 | Jan 2020 | US | |
| 62963897 | Jan 2020 | US |
