The present disclosure provides compositions and methods for using fixed biological samples in partition-based assays. In at least one embodiment, the disclosure provides a composition comprising a fixed biological sample and an un-fixing agent contained in a partition, such as a discrete droplet. In some embodiments, the disclosure provides un-fixing agent compounds capable of catalytically cleaving crosslinks in fixed biological samples, particularly crosslinked nucleic acids, such as RNA.
C07D 213/04 - Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
C07D 239/24 - Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
C12N 9/48 - Hydrolases (3.) acting on peptide bonds, e.g. thromboplastin, leucine aminopeptidase (3.4)
G01N 33/50 - Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
Provided herein are systems and methods for drug screening single cells from a sample. A method for drug screening may comprise treating various single cell samples with different drugs or drug combinations, labeling of treated cells with a labeling agent comprising a sample barcode, and processing the treated, labeled cells to generate nucleic acid libraries for sequencing. One or more processes of the methods described herein may be performed within a partition, such as a droplet or well.
C12Q 1/6876 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
C12Q 1/25 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups
C12Q 1/6811 - Selection methods for production or design of target specific oligonucleotides or binding molecules
C12Q 1/6816 - Hybridisation assays characterised by the detection means
Provided in some aspects are methods for light-controlled in situ surface patterning of a substrate. Compositions such as nucleic acid arrays produced by the methods are also disclosed. Further provided in some aspects are methods for light-controlled in situ refinement of nucleic acid array feature boundaries. In some embodiments, a method disclosed herein comprises using photoresist for photocontrollable removal, blocking, and/or inactivation of nucleic acid molecules at feature boundaries. A large diversity of barcodes can be created in molecules on the substrate via sequential rounds of light exposure, hybridization, ligation, and further interceded and/or followed by one or more rounds of photocontrollable refinement of array feature boundaries, to improve spatial array resolution and sensitivity.
The present disclosure relates in some aspects to methods and compositions for in situ analysis involving catalytic de-crosslinking of biological samples.
Systems, and their methods of use, for sorting or separating magnetic particles are provided. A system having a magnetic module with features that mate with voids in a flow module exerts a magnetic field on magnetic particles to separate particles.
The present disclosure relates in some aspects to recombinant polymerases, variant PumA1 polymerases, and compositions thereof. Also provided herein are methods of using the recombinant polymerases and/or variant PumA1 polymerases for nucleic acid amplification (e.g., rolling circle amplification). In some aspects, the compositions and methods disclosed herein provide more robust amplification (e.g., RCA) reactions for improved in vitro and in situ analysis.
The present disclosure generally relates to methods and systems for engineering antibodies, and antigen-binding fragments thereof, to have altered characteristics. The present disclosure also provides a high-throughput method useful for identifying the engineered antibodies, or antigen-binding fragments thereof, that acquired the altered characteristics having performed the methods. These methods and systems have implications, for example, in the rapid development of biotherapeutics having desired and/or improved, e.g., affinity, specificity and/or activity-related, characteristics.
The present disclosure relates to materials and methods for spatially analyzing nucleic acids fragmented with a transposase enzyme in a biological sample.
A method for identifying framework regions and complementarity-determining regions in an amino acid sequence. The amino acid sequence is received. A plurality of candidate start positions is identified within the amino acid sequence for a start position for a selected region of interest. A score is generated for each candidate start position of the plurality of candidate start positions via analysis of a motif window that begins at each candidate start position. The start position for the selected region of interest is identified based on a candidate start position of the plurality of candidate start positions having a highest score.
G16B 30/00 - ICT specially adapted for sequence analysis involving nucleotides or amino acids
G16B 40/00 - ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
A sample holder includes a first member featuring a first retaining mechanism configured to retain a first substrate that includes a sample, a second member featuring a second retaining mechanism configured to retain a second substrate that includes a reagent medium, and an alignment mechanism connected to at least one of the first and second members, and configured to align the first and second members such that the sample contacts at least a portion of the reagent medium when the first and second members are aligned.
The present disclosure relates in some aspects to recombinant Bacillus phage AP50 polymerases, modified AP50 polymerases, and compositions thereof. Also provided are methods of using the recombinant and/or modified AP50 polymerases for nucleic acid amplification (e.g., rolling circle amplification). In some aspects, the compositions and methods disclosed herein provide more robust amplification (e.g., RCA) reactions for improved in vitro and in situ analysis.
Methods and compositions for performing base-by-base sequencing in situ in a cell or tissue sample that minimize optical crowding are described. In some embodiments, a sequencing primer hybridizes to a priming site 3′ to an identifier sequence (e.g., a barcode sequence) in the sample such that the sequencing primer can be extended by a polymerase in a base-by-base fashion using the identifier sequence as a template. The sample can be contacted with nucleotides in a cyclic series of nucleotide incorporation or binding steps, and signals indicative of the incorporation or binding events can be detected to generate signal code sequences comprising a series of signal codes (corresponding to signals (ON signals), absence of signals (OFF signals), or a combination thereof) detected in the sequential cycles. Decoding of the identifier sequences based at least in part of the signal code sequences can be used to detect and locate the corresponding analytes.
Provided in some aspects are methods of patterning a surface in situ for producing an array on the surface, for example, by partitioning of beads comprising oligonucleotides into spatially predefined regions, to generate unique DNA sequences in spatial positions in the array. Compositions such as nucleic acid arrays produced by the methods are also disclosed.
Methods and systems for sample preparation techniques that allow amplification (e.g., whole genome amplification) and sequencing of chromatin accessible regions of single cells are provided. The methods and systems generally operate by forming or providing partitions (e.g., droplets) including a single biological particle and a single bead comprising a barcoded oligonucleotide. The preparation of barcoded next-generation sequencing libraries prepared from a single cell is facilitated by the transposon-mediated transposition and fragmentation of a target nucleic acid sequence. The methods and systems may be configured to allow the implementation of single-operation or multi-operation chemical and/or biochemical processing within the partitions.
Provided herein are methods of improving sensitivity of spatial detection of an analyte in a biological sample using splint oligonucleotides, circularized second strands, and rolling circle amplification. For example, provided herein are methods of improving sensitivity of spatial detection of an analyte in a biological sample where a splint oligonucleotide hybridizes to a second strand; the second strand is ligated together thereby creating a circularized second strand, rolling circle amplification of the circularized second strand results in generation of an amplified second strand, and all or part of the sequence of the amplified second strand is determined and used to spatially detect the analyte in the biological sample.
Aspects of the present invention include analyzing nucleic acids from single cells using methods that include using tagged polynucleotides containing multiplex identifier sequences.
This disclosure relates to compositions and methods for three-dimensional spatial profiling of analytes in a biological sample. The methods include use of a hydrogel comprising one or more polymers that include a phenol moiety, an azide moiety, or an alkyne moiety.
A method of analyzing single biological particles, such as cells or nuclei, that maintains the single biological particles in a state of relative isolation during decrosslinking and subsequent processing is provided. In the case of cells, the method prevents cellular analytes from each individual cell from leaving the cell site and diffusing toward adjacent cells, while permitting transmission of a decrosslinking agent, followed by processing of the cellular analytes from the individual cells by barcoding and/or imaging of the cellular analytes.
Provided herein are methods of determining a location of an analyte in a biological sample, and systems for performing these methods. In particular, the methods and systems comprise decreasing the binding events of an analyte from a biological sample to capture probes that surround a biological sample positioned on a substrate.
The present disclosure relates in some aspects to methods for manufacturing molecular arrays using a hybrid approach comprising microfluidics-based delivery and photolithography-guided oligonucleotide hybridization and ligation. In particular, the molecular arrays can be used for determining spatial patterns of abundance and/or expression of a biological target in a sample.
The present disclosure relates in some aspects to methods and compositions for processes for inverting oligonucleotide molecules in an in situ synthesized array, including reversing the orientation of the oligonucleotide molecules with respect to the array substrate from 3′-immobilized to 5′-immobilized.
Provided in some aspects are methods for light-controlled in situ surface patterning of a substrate comprising a step of blocking or ablating oligonucleotide molecules in a boundary region separating a plurality of spot regions, and attaching oligonucleotides to oligonucleotide molecules in a first one or more of the spot regions.
G03F 7/11 - Photosensitive materials - characterised by structural details, e.g. supports, auxiliary layers having cover layers or intermediate layers, e.g. subbing layers
The present disclosure relates generally to compositions, methods, kits, partitions and systems for high-throughput microfluidic identification of antigen-binding molecules and antigen binding molecule epitope mapping. Particularly, the compositions, methods, kits, partitions and systems of the disclosure relate to the characterization of cell-expressed antigen-binding molecules (e.g., antibodies) produced by a population of cells, such as B cells, that may have different binding and epitope specificity to a target antigen of interest and/or with enhanced activity.
The present disclosure provides methods, compositions and systems for analyzing individual cells or cell populations through a partitioned analysis of contents of individual cells or cell populations, such as cancer cells and cells of the immune system. Individual cells or cell populations may be co-partitioned with processing reagents for accessing cellular contents, and for uniquely identifying the content of a given cell or cell population, and subsequently analyzing the content of the cell and characterizing it as having derived from an individual cell or cell population, including analysis and characterization of nucleic acid(s) from the cell through sequencing.
The present disclosure in some aspects relates to methods and compositions for improved detection and quantification of one or more analytes present in a biological sample. In some aspects, the methods and compositions provided herein address issues associated with the heterogeneity of rolling circle amplification (RCA) products during in situ analyses. In some aspects, the methods and compositions disclosed herein provide more homogenous RCA products for improved sample imaging.
Provided in some aspects are methods for light-controlled in situ surface patterning of a substrate using a minimal mask scheme. Systems and kits employing the methods are also disclosed. In some embodiments, a method disclosed herein comprises using photomasks and photoresist for photocontrollable hybridization and/or ligation of nucleic acid molecules, wherein photoresist removal allows hybridization and/or ligation of nucleic acid molecules at the exposed area. A large diversity of barcodes can be created in molecules on the substrate via sequential rounds of light exposure, hybridization, and ligation.
C12Q 1/6874 - Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation [SBH]
C12Q 1/6876 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
G03F 7/00 - Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printed surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
32.
METHODS AND SYSTEMS FOR CHARACTERIZING ANALYTES FROM INDIVIDUAL CELLS OR CELL POPULATIONS
The present disclosure provides methods of processing or analyzing a sample. A method for processing a sample may comprise hybridizing a probe molecule to a target region of a nucleic acid molecule (e.g., a ribonucleic acid (RNA) molecule), barcoding the probe-nucleic acid molecule complex, and performing extension, denaturation, and amplification processes. A method for processing a sample may comprise hybridizing first and second probes to adjacent or non-adjacent target regions of a nucleic acid molecule (e.g., an RNA molecule), linking the first and second probes to provide a probe-linked nucleic acid molecule, and barcoding the probe-linked nucleic acid molecule. One or more processes of the methods described herein may be performed within a partition, such as a droplet or well. One or more processes of the methods described herein may be performed on a cell, such as a permeabilized cell.
G01N 33/532 - Production of labelled immunochemicals
C07K 14/74 - Major histocompatibility complex (MHC)
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12N 15/11 - DNA or RNA fragments; Modified forms thereof
C12N 15/85 - Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
C12Q 1/6804 - Nucleic acid analysis using immunogens
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/6818 - Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
C12Q 1/6827 - Hybridisation assays for detection of mutation or polymorphism
C12Q 1/6881 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
G01N 33/50 - Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
Provided herein are methods for preparing biological samples for spatial proteomic analysis, methods of determining a location of a protein analyte in a biological sample, and methods of determining a location of a protein analyte and a nucleic acid analyte in a biological sample.
The present disclosure provides methods and systems for nucleic acid preparation and/or analysis. Nucleic acids may be derived from one or more cells. Nucleic acid preparation may comprise generating nucleic acid molecules of varying lengths. Nucleic acid analysis may comprise identifying nucleic acid sequence information with nucleosome position information.
Systems and methods for analyzing a tissue sample are provided. A set of images of the tissue on a substrate comprising capture spots is obtained. Each image is an emission image of the tissue upon excitation of the tissue at a corresponding excitation wavelength for a respective detectable marker. A first spatial dataset is acquired including, for each detectable marker for each respective image, a measured intensity of each capture spot indexed by a representative spatial barcode in a plurality of spatial barcodes. A second spatial dataset is acquired including nucleic acid quantification data representing a number of nucleic acid molecules originating from the tissue on the substrate and localized to each capture spot by an associated spatial barcode. At least a portion of the first and second spatial datasets co-registered to each other by the spatial barcodes are spatially displayed, on a display.
The present disclosure generally relates to compositions and methods for secreted analyte detection and analysis. The analytes detected and analyzed are mapped to a cell or nucleus having secreted the analyte and are useful to characterize the cell or nucleus. The ability to understand analyte secretion events has implications for the improvement of drug delivery, cell profiling, and diagnostic development.
Methods and systems for selecting a cell of interest based on immune cell data are disclosed. For example, a method may comprise obtaining a single cell or spatial dataset, wherein the single cell or spatial dataset comprises a dataset of immune cell receptors, antibodies, or fragments thereof from a sample; identifying a clonotype group in the single cell or spatial dataset;
Methods and systems for selecting a cell of interest based on immune cell data are disclosed. For example, a method may comprise obtaining a single cell or spatial dataset, wherein the single cell or spatial dataset comprises a dataset of immune cell receptors, antibodies, or fragments thereof from a sample; identifying a clonotype group in the single cell or spatial dataset;
selecting a schema to visualize selected amino acids in the clonotype group based on positions or chemical identity of the selected amino acids; visualizing the selected amino acids in the clonotype group in a graphic representation according to the schema; and selecting a cell of interest from the clonotype group based on a pre-defined criterion using the graphic representation.
The present disclosure provides systems and methods for processing or analyzing a sample. Methods may include generating supports (e.g., beads) comprising barcode molecules coupled thereto. A support comprising barcode molecules may be useful for analyzing or processing one or more analytes such as nucleic acid molecules, proteins, and/or perturbation agents.
G01N 33/532 - Production of labelled immunochemicals
C07K 14/74 - Major histocompatibility complex (MHC)
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12N 15/11 - DNA or RNA fragments; Modified forms thereof
C12N 15/85 - Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
C12Q 1/6804 - Nucleic acid analysis using immunogens
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/6818 - Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
C12Q 1/6827 - Hybridisation assays for detection of mutation or polymorphism
C12Q 1/6881 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
G01N 33/50 - Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
An assembly includes a stage body, a cam plate disposed on the body, and a sample positioning plate having a sample positioning surface configured to receive a sample device. The surface has first, second, and third raised portions. The second raised portion is disposed between the first and third raised portions. The first, second, and third raised portions are configured to contact the sample device. The assembly includes a riser module disposed within the stage body. The riser module is coupled to the plate and the body. The assembly includes a lid configured to be coupled to the cam plate. The lid has a coupled configuration and an uncoupled configuration such that, when in the coupled configuration, a recess is formed by the lid and the plate. The assembly includes one or more light sources disposed in the cam plate that are configured to direct light within the recess.
The present disclosure relates in some aspects to methods and compositions for analyzing a target nucleic acid in a biological sample. In some aspects, the methods disclosed herein promote hybridization of polynucleotides to target nucleic acids and/or splints for enhanced ligation efficiency and/or enhanced hybridization efficiency in situ in a biological sample. In some aspects, the presence, amount, and/or identity of a target nucleic acid is analyzed in situ. Also provided are compositions and kits for use in accordance with the methods.
The present disclosure generally relates to methods and compositions for in situ analysis or detection of analytes in a biological sample. More specifically, the present disclosure relates to methods for reducing autofluorescence in tissue samples, methods for analyzing tissue samples, and compounds for use in the same. The methods and compounds of the present disclosure may be especially suitable for analytical methods employing fluorescence in situ hybridization techniques over multiple cycles of imaging.
Provided herein are methods, compositions, and kits for determining a location of a target analyte in a biological sample that include the use of terminal deoxynucleotidyl transferase.
Various embodiments of the present disclosure disclose a system for forming a sealed chamber for preparing samples therein. In particular, various embodiments present an assembly that includes a sample device and a lid for forming a sealed chamber that can be used for, among other things, incubating samples therein. When the lid engages with the sample device, a plurality of snap joint elements of the lid engage with a respective plurality of snap joint elements to form a seal between the lid and the sample device.
A sample holder is provided. The sample holder includes a first member including a first retaining mechanism configured to retain a first substrate comprising a sample. The first retaining mechanism configured to retain the first substrate disposed in a first plane. The sample holder further includes a second member including a second retaining mechanism configured to retain a second substrate including a reagent medium disposed in a second plane. The sample holder further includes an alignment mechanism connected to one or both of the first member and the second member. The alignment mechanism configured to align the first and second members along the first plane and/or the second plane such that the sample contacts at least a portion of the reagent medium when the first and second members are aligned and within a threshold distance along an axis orthogonal to the second plane.
The present disclosure relates in some aspects to methods and compositions for in situ analysis involving catalytic de-crosslinking of biological samples.
The present invention is directed to methods, compositions and systems for capturing and analyzing sequence information contained in targeted regions of a genome. Such targeted regions may include exomes, partial exomes, introns, combinations of exonic and intronic regions, genes, panels of genes, and any other subsets of a whole genome that may be of interest.
A substrate holder includes a base configured to receive a substrate; a cover configured to mateably engage with the base, the cover defining an opening formed by inner sidewalls; and a removable insert defining a surface, the removable insert being configured to be received within the opening of the cover. The removable insert includes a gasket; a projection coupled to the gasket; and at least two insert tabs extending from opposite sides of the removable insert, each insert tab being configured to engage with at least one of the inner sidewalls forming the opening of the cover.
The present disclosure generally relates to methods and compositions for detecting a plurality of molecules of one or more analytes in a sample. In some aspects, the present disclosure relates to methods for determining the locations and identities of analytes in a biological sample using detectably labeled probes comprising detectable labels with different signal emission lifetimes. In some aspects, the present disclosure relates to methods for identifying the detectable labels of the detectably labeled probes using time-gated detection. The methods herein have particular applicability in the detection of identifier sequences (e.g., analyte sequences or barcode sequences) in situ in a biological sample, including those using sequential cycles of detectably labeled probe hybridization to decode the identifier sequences.
The present disclosure generally relates to methods and compositions for in situ analysis or detection of analytes in a sample. More specifically, the present disclosure relates to methods for reducing autofluorescence in tissue samples, methods for analyzing biological samples, and compounds for use in the same. The methods and compounds of the present disclosure may be especially suitable for analytical methods employing fluorescence in situ hybridization techniques over multiple cycles of imaging.
The present disclosure relates in some aspects to methods and compositions for in situ analysis using nucleic acid complexes comprising a detectable label and a moiety that can be activated to extinguish signals of the detectable label. In some embodiments, the activatable moiety comprises a photosensitizer. The nucleic acid complexes may allow for improved detection and decreased signal carryover between detection cycles.
G01N 21/35 - Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
The present disclosure relates to methods, compositions, systems, and kits for detecting and analyzing the glycosylation of healthy and diseased cells and protein-specific glycosylation patterns using single-cell profiling methodologies.
In some aspects, the present disclosure relates to methods for reducing the detection of false positive events during analysis of a biological sample. In some aspects, the method comprises use of a decoy oligonucleotide that can hybridize to a probe or to a target nucleic acid. The methods herein have particular applicability in reducing the detection of false positive events and in combining hybridization and ligation reactions into a single step. Also provided are kits comprising probes for use in such methods.
The present disclosure relates in some aspects to methods and compositions for analyzing biological samples involving active stripping of detectably labeled probes from hybridization complexes such as rolling circle amplification products (RCPs). In some embodiments, the present application provides a method wherein double-stranded hybridized complexes are dissociated by DNA helicases. In some embodiments, single-stranded binding proteins facilitate the dissociation and prevent hybridization of the dissociated strands.
The present disclosure relates in some aspects to methods for analyzing antigen receptor transcripts in a biological sample. In some aspects, nucleic acid molecules are generated from V(D)J transcripts in situ in the biological sample to enrich molecules comprising V(D)J joins. In some aspects, the presence, amount, and/or identity of a plurality of V(D)J transcripts are analyzed in situ. Also provided are oligonucleotides, sets of oligonucleotides, compositions, and kits for use in accordance with the methods.
The present disclosure relates in some aspects to methods and compositions for manufacturing molecular arrays using a hybrid approach comprising using non-contact printing (e.g., using inkjet printing, slot die coating, and/or blade coating) and photolithography-guided oligonucleotide hybridization and ligation. In particular, the molecular arrays can be used for determining spatial patterns of abundance and/or expression of a biological target in a sample.
Provided herein are systems and methods for processing biomolecules (e.g., nucleic acid molecules, proteins) from a sample. A method for processing biomolecules may comprise hybridizing a probe molecule to a target region of a nucleic acid molecule (e.g., a ribonucleic acid (RNA) molecule) and barcoding the probe-nucleic acid molecule complex or derivatives thereof. Such a method can comprise performing a nucleic acid reaction, e.g., extension, denaturation, and amplification. A method for processing a sample may comprise hybridizing probes to (i) target regions of a nucleic acid molecule (e.g., RNA molecule) and (ii) a reporter oligonucleotide of a feature binding group, and barcoding the probe-associated molecules. One or more processes of the methods described herein may be performed within a partition, such as a droplet or well.
G01N 33/532 - Production of labelled immunochemicals
C07K 14/74 - Major histocompatibility complex (MHC)
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12N 15/11 - DNA or RNA fragments; Modified forms thereof
C12N 15/85 - Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
C12Q 1/6804 - Nucleic acid analysis using immunogens
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/6818 - Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
C12Q 1/6827 - Hybridisation assays for detection of mutation or polymorphism
C12Q 1/6881 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
G01N 33/50 - Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
Devices, methods, and systems for generating droplets are provided. The devices, methods, and systems are designed to maximize droplet recovery, e.g., from a collection reservoir.
Provided herein are methods of identifying a location of an analyte in a biological sample using padlock oligonucleotides and rolling circle amplification. For example, provided herein are methods for identifying a location of an analyte in a biological sample where (i) a padlock oligonucleotide hybridizes to an analyte and is ligated thereby creating a circularized padlock oligonucleotide, (ii) rolling circle amplification of the circularized padlock oligonucleotide results in generation of an amplified circularized padlock oligonucleotide, and (iii) a signal corresponding to the amplified circularized padlock oligonucleotide is detected and used to identify the location of the analyte in the biological sample. Also provided are methods for identifying whether a treatment of a biological sample facilitates the release and subsequent detection of an analyte from the biological sample using padlock oligonucleotides and rolling circle amplification.
The present disclosure relates to materials and methods for spatially analyzing nucleic acids that have been fragmented with a transposase enzyme, alone or in combination with other types of analytes.
C12Q 1/6881 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
C12Q 1/6874 - Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation [SBH]
The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing.
C12Q 1/6874 - Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation [SBH]
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/683 - Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
C12Q 1/6804 - Nucleic acid analysis using immunogens
67.
MULTIPLEX CAPTURE OF GENE AND PROTEIN EXPRESSION FROM A BIOLOGICAL SAMPLE
Provided herein are methods, compositions, and kits for preparing biological samples for multiplex spatial gene expression and proteomic analysis, such as determining a location of a nucleic acid analyte and a protein analyte in a biological sample.
This disclosure provides methods and compositions for sample processing, particularly for sequencing applications. Included within this disclosure are bead compositions, such as diverse libraries of beads attached to large numbers of oligonucleotides containing barcodes. Often, the beads provides herein are degradable. For example, they may contain disulfide bonds that are susceptible to reducing agents. The methods provided herein include methods of making libraries of barcoded beads as well as methods of combining the beads with a sample, such as by using a microfluidic device.
The present disclosure provides methods and systems for cell and bead processing or analysis. A method for processing a cell or bead may comprise subjecting a bead to conditions sufficient to change a first characteristic or set of characteristics (e.g., cell or bead size). Such a method may further comprise subjecting the cell or bead to conditions sufficient to change a second characteristic or set of characteristics. In some cases, crosslinks may be formed within the cell or bead.
The present disclosure relates in some aspects to methods of contacting a biological sample (e.g., fresh or frozen tissue sample) with peptide-loaded detection complexes. In some aspects, the peptide-loaded detection complexes comprise antigen presenting molecule monomers or multimers that bind to antigens, reporter oligonucleotides that correspond to the antigen/antigen presenting molecule combination. In some aspects, the peptide-loaded detection complexes are tetramers. In some aspects, the tetramers bound to T cell receptors of T cells may be detected in situ. In some aspects, the tetramers bound to TCRs may be detected on an in situ platform and/or using an array comprising spatially barcoded capture agents. In some embodiments, the method comprises contacting a plurality of peptide-loaded detection complexes to clonal populations of T cells comprising TCRs of different antigen specificities.
Disclosed herein are methods of amplifying an analyte in a biological sample using a bridging oligonucleotide that hybridizes to a captured analyte. The methods disclosed herein include steps of (a) contacting a biological sample with a substrate having capture probes comprising a capture domain and a spatial barcode; (b) hybridizing the analyte to the capture domain; and (c) contacting the analyte to a bridging oligonucleotide comprising (i) a capture-probe-binding sequence, and (ii) an analyte-binding sequence; (d) extending the bridging oligonucleotide; and (e) determining (i) all or a part of the sequence of the analyte, or a complement thereof, and (ii) the spatial barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to determine the location of the analyte in the biological sample.
The present disclosure relates in some aspects to methods and compositions for light-controlled in situ surface patterning of a substrate using a Click chemistry reaction. In some embodiments, a method disclosed herein comprises a copper-catalyzed alkyne-azide (CuAAC) click chemistry reaction for photocontrollable ligation of nucleic acid molecules. A large diversity of barcodes can be created in molecules on the substrate via sequential rounds of light exposure, hybridization, and ligation.
Provided herein are reaction mixtures, compositions, systems, methods, and kits for assessing enzymatic activity. Aspects of the disclosure include use of a reactant barcoded oligonucleotide (RBO) construct comprising a first oligonucleotide comprising a reaction barcode sequence and a first linker that connects the first oligonucleotide to a first reactant, an amplification construct comprising a second oligonucleotide that is complementary to at least a portion of the first oligonucleotide and a second linker that connects the second oligonucleotide to a second reactant, and a blocking construct, comprising a third oligonucleotide that is complementary to at least a portion of the first oligonucleotide. A rate of reaction product generation may be measured to determine an enzymatic activity of a given enzyme.
The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing.
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C40B 20/04 - Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
C12Q 1/6804 - Nucleic acid analysis using immunogens
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C40B 50/16 - Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support involving encoding steps
C12Q 1/6874 - Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation [SBH]
C12Q 1/683 - Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
75.
METHODS AND COMPOSITIONS FOR PROBE DETECTION AND READOUT SIGNAL GENERATION
In some aspects disclosed herein are methods and compositions for detecting an analyte such as a target nucleic acid in a biological sample, said method comprising generating and analyzing a detectable signal associated with the target nucleic acid and a separate signal associated with a region of interest in the target nucleic acid.
C12Q 1/6881 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
Provided herein are methods for capturing an exogenous probe and/or a capture handle sequence to a capture domain of a capture probe. Compositions, systems, and kits also are disclosed. In some instances, the methods include detecting an exogenous nucleic acid in a biological sample by hybridizing a first exogenous probe oligonucleotide and a second exogenous probe oligonucleotide to an exogenous nucleic acid in a biological sample on a first substrate, coupling the exogenous probe oligonucleotides, and capturing the coupled exogenous probe on a spatial array.
C12Q 1/70 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
C12Q 1/6818 - Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
77.
METHODS, COMPOSITIONS, AND SYSTEMS FOR IMPROVED IN SITU DETECTION OF ANALYTES AND SPATIAL ANALYSIS
Provided herein are methods, compositions, and systems for improved in situ detection of analytes and spatial analysis using, e.g., a sequencing readout.
Provided herein are methods and systems for analysis of one or more single cells. The disclosed methods may comprise barcoding analytes from cell types of interest, while eliminating undesired cell types from analysis. Barcoding may comprise the use of a targeting agent. A targeting agent may be attached to an inhibitor or an activator, thereby inhibiting or activating analyte barcoding.
Disclosed are methods, compositions and kits for contacting a sample containing a biological particle with a catalyst associated with or attached to a support. The biological particle may be cells and/or nuclei. The catalyst may be an enzyme configured to digest an extracellular molecule, such as an extracellular biological molecule, including extracellular nucleic acid molecules. In some examples, the biological particle is an aggregate of cells that is processed to single cells with a nuclease that is attached to a bead support. The bead and nuclease may subsequently be removed from the system. The single cells that result from the method can be used in single cell-based droplet systems for obtaining genome or transcriptome profiles of single cells.
Provided herein are methods for analyzing immune cell infiltration in a cancer stromal region of a biological sample obtained from a subject using machine learning modules. For example, the methods may include (a) identifying a cancerous region or an analyte associated with the cancerous region in the biological sample; (b) identifying a stromal region or an analyte associated with the stromal region in the biological sample; (c) identifying one or more immune cells or an analyte associated with an immune cell in one or more locations in the biological sample; and (d) using (i) the identified cancerous and stromal regions or associated analytes thereof in the biological sample and (ii) the identified one or more immune cells or associated analytes thereof to analyze immune cell infiltration in the cancer stromal region of the biological sample obtained from the subject.
C12Q 1/6886 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
G16H 50/20 - ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for computer-aided diagnosis, e.g. based on medical expert systems
81.
Methods and systems for multiplexing of droplet based assays
Compositions, methods, and systems are provided for sample preparation techniques and sequencing of macromolecular constituents derived from a cell (i.e., a cell bead) in a multiplexed reaction. Using the compositions, systems, and methods disclosed herein, the association of the macromolecular constituents with the biological particle from which they are derived and the association of the cell bead with the cell bead sample from which they are derived is maintained.
Provided herein are systems and methods for processing biomolecules (e.g., nucleic acid molecules, proteins) from a sample. A method for processing biomolecules may comprise hybridizing a probe molecule to a target region of a nucleic acid molecule (e.g., a ribonucleic acid (RNA) molecule) and barcoding the probe-nucleic acid molecule complex or derivatives thereof. Such a method can comprise performing a nucleic acid reaction, e.g., extension, denaturation, and amplification. A method for processing a sample may comprise hybridizing probes to (i) target regions of a nucleic acid molecule (e.g., RNA molecule) and (ii) a reporter oligonucleotide of a feature binding group, and barcoding the probe-associated molecules. One or more processes of the methods described herein may be performed within a partition, such as a droplet or well.
The invention provides devices for forming droplets and concentrating droplets, and methods of their use. During operation of the devices, droplets are generated using two liquid phases. Once droplet formation is complete excess continuous phase is removed by the use of one or more pressure differentials.
The present disclosure relates generally to the field of immunology, and particularly relates to compositions, methods, and systems for the analysis and generation of antigen-binding molecules produced by immune cells obtained from tumor samples (e.g., antibodies produced by B cells obtained from tumor samples or TCRs produced by T cells obtained from tumor samples), using single-cell immune profiling methodologies, so as to produce recombinant antigen-binding molecules (e.g., antibodies, TCRs) with desired properties.
A discrete attribute value dataset is obtained that is associated with a plurality of probe spots each assigned a different probe spot barcode. The dataset comprises spatial projections, each comprising images of a biological sample. Each image includes a corresponding plurality of discrete attribute values for the probe spots. Each such value is associated with a probe spot in the plurality of probes spots based on the probe spot barcodes. The dataset is clustered using the discrete attribute values, or dimension reduction components thereof, for a plurality of loci at each respective probe spot across the images of the projections thereby assigning each probe spot to a cluster in a plurality of clusters. Morphological patterns are identified from the spatial arrangement of the probe spots in the various clusters.
G06V 10/762 - Arrangements for image or video recognition or understanding using pattern recognition or machine learning using clustering, e.g. of similar faces in social networks
G16B 15/00 - ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
C12Q 1/6876 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
C12Q 1/6811 - Selection methods for production or design of target specific oligonucleotides or binding molecules
C12Q 1/25 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups
C12Q 1/6816 - Hybridisation assays characterised by the detection means
The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing and analyte characterization from a single cell. Such polynucleotide processing may be useful for a variety of applications. The compositions, methods, systems, and devices disclosed herein generally describe barcoded oligonucleotides, which can be bound to a bead, such as a gel bead, useful for characterizing one or more analytes.
C40B 30/04 - Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
C07K 14/74 - Major histocompatibility complex (MHC)
C12N 15/85 - Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
G01N 33/532 - Production of labelled immunochemicals
G01N 33/543 - Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
G01N 33/58 - Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
C12Q 1/6818 - Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
C12Q 1/6827 - Hybridisation assays for detection of mutation or polymorphism
C12Q 1/6881 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
C12N 15/11 - DNA or RNA fragments; Modified forms thereof
G01N 33/569 - Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
89.
METHODS AND COMPOSITIONS FOR ANALYZING ANTIGEN BINDING MOLECULES
Provided herein are methods and systems for measuring secreted antibodies or other analytes from single cells. The methods disclosed herein include the use of capture agents and reporter agents that bind to secreted antibodies, wherein the capture agent and/or the reporter agent can be analyte-specific and/or barcoded, and beads in partitions (e.g., droplets or wells) to measure secreted antibodies on a single cell basis. Further described herein are methods comprising the use of hydrogel-encapsulated cells (e.g., cell beads) to measure secreted and/or cellular analytes from single cells.
Provided herein are methods of identifying a location of an RNA in a sample that include: (a) contacting the sample with an array comprising capture probes, where a capture probe comprises a capture domain and a spatial barcode; (b) releasing the RNA from the sample; (c) extending a 3′ end of the capture probe using the capture domain-bound RNA as a template; (d) generating nick(s) in the extended capture probe-hybridized RNA and performing random-primed DNA synthesis; (e) performing end repair on the second strand DNA molecule; (f) adding a single adenosine nucleotide to the 3′ end of the extended capture probe; (g) ligating a double-stranded sequencing adaptor to the double-stranded DNA product; and (h) determining all or a part of the sequence of the RNA, and the sequence of the spatial barcode, or complements thereof, and using the determined sequences to identify the location of the RNA in the sample.
Provided herein are methods of improving sensitivity of spatial detection of an analyte in a biological sample using splint oligonucleotides, circularized second strands, and rolling circle amplification. For example, provided herein are methods of improving sensitivity of spatial detection of an analyte in a biological sample where a splint oligonucleotide hybridizes to a second strand; the second strand is ligated together thereby creating a circularized second strand, rolling circle amplification of the circularized second strand results in generation of an amplified second strand, and all or part of the sequence of the amplified second strand is determined and used to spatially detect the analyte in the biological sample.
The present disclosure relates in some aspects to methods for analyzing a target RNA in a biological sample, such as detection of a sequence of interest in an RNA. In some aspects, provided herein are methods of using an RNase such as RNase H to provide in situ RNA detection methods having high sensitivity, efficiency, and/or specificity.
Systems and methods for inferring a status of a cell population are provided. Described techniques allow deconvolving a first clonal population comprising a first plurality of cells of a species, wherein nucleic acid sequence reads from each cell in the first plurality of cells are obtained. The nucleic acid sequence reads are mapped into bins representing portions of a reference genome, and a pattern of sequence read counts for each cell across the multiple bins is used to assign a cell to a group, thereby inferring a mitotic status of the cell. The assignment of nucleic acid sequence reads into bins is also be used for segregating cells into classes based on a status of a certain biological marker in each cell. Comparison of sequence read counts for a subset of bins across the cell classes allows evaluating effect of a compound on a cell status.
G16B 30/00 - ICT specially adapted for sequence analysis involving nucleotides or amino acids
G16H 10/40 - ICT specially adapted for the handling or processing of patient-related medical or healthcare data for data related to laboratory analysis, e.g. patient specimen analysis
G16H 20/00 - ICT specially adapted for therapies or health-improving plans, e.g. for handling prescriptions, for steering therapy or for monitoring patient compliance
The present disclosure relates generally to engineered nucleic acid processing enzymes and derivatives thereof, compositions and kits comprising the same; and methods of generating and using the same.
The present disclosure relates in some aspects to methods and compositions for analyzing a biological sample, comprising performing rolling circle amplification of a circRNA in situ in a biological sample, thereby generating an RCA product comprising multiple complementary copies of the circRNA and detecting the RCA product in situ at a spatially localized position in the biological sample.
A dataset is obtained comprising data blocks, each representing a different characteristic, for a plurality of cells across a plurality of bins, each bin representing a different portion of a reference sequence. Cells are clustered on one such characteristic across the bins thereby forming a tree that includes root, intermediate, and terminal nodes, where the cells are terminal nodes and intermediate nodes have daughter nodes, themselves being intermediate nodes or a cell. A subset of the tree is displayed that includes the root and leaves, each leaf representing an intermediate node or a cell. A heat map of the characteristic is also displayed, the map including a segment for each leaf, across the bins. When a segment represents an intermediate node, it is an average of the characteristic across daughters of the node. Graphs of characteristics for the root across the bins are also displayed.
The present application provides methods for detecting a target nucleic acid molecule in a sample comprising contacting said sample with a ligatable probe comprising one or more parts and allowing said probe to hybridise to the target nucleic acid molecule, ligating any probe which has hybridised to the target nucleic acid molecule, amplifying the ligated probe, and detecting the amplification product, thereby to detect the target nucleic acid molecule, wherein said probes comprise at least one ribonucleotide at or near to a ligation site and/or wherein the probe or a probe part comprises an additional sequence 5′ to a target-specific binding site which is not hybridised to the target nucleic acid molecule upon hybridisation of the probe to the target nucleic acid molecule and forms a 5′ flap containing one or more nucleotides at its 3′ end that is cleaved prior to ligation, and methods of synthesising a DNA molecule with Phi29 DNA polymerase using a template nucleic acid molecule comprising at least one ribonucleotide. Probes for use in the detection methods are provided.
The disclosure provides methods for separating and/or purifying one or more molecules released from one or more fluid compartments or partitions, such as one or more droplets. Molecules can be released from a fluid compartment(s) and bound to supports that can be isolated via any suitable method, including example methods described herein. The disclosure also provides devices that can aid in isolating supports bound to molecules.
patents
