The present disclosure relates generally to compositions, methods and systems for characterizing a biological particle, cell or cell nucleus, modified to include an exogenous insert, e.g., exogenous nucleic acids. The characterization may identify the insertion site of the exogenous nucleic acids in the biological particle's endogenous, e.g., genomic, nucleic acid sequences. Such characterization may be useful in the development of safer, more effective, modified cell biotherapeutics, e.g., cells modified to express chimeric antigen receptors.
The present disclosure relates generally to compositions, methods, and systems for the characterization of antigen-binding molecules (e.g., antibodies) in biological samples. This characterization permits the identification of the ABM that bind to regions of interest, or the mapping of ABMs according to their binding to specific regions of interest, of an antigen. Understanding the binding characteristics of ABMs at a region of interest level may facilitate the identification and production of immunotherapeutic molecules having desired properties.
Crosslinked fluorocarbon surface coating, kits therefor, microfluidic devices having crosslinked fluoropolymer surface coatings, and methods of use for droplet generation are provided. The kits, devices, and their methods have a droplet source region that is coated with a crosslinked fluoropolymer surface coating produced by the reaction of a fluorocarbon silane and a fluorocarbon polyol.
C08G 65/00 - Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
C08G 65/336 - Polymers modified by chemical after-treatment with organic compounds containing silicon
C09D 171/00 - Coating compositions based on polyethers obtained by reactions forming an ether link in the main chain; Coating compositions based on derivatives of such polymers
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.
Provided herein are methods, compositions, and kits for reducing capture of analytes from a biological sample on a spatial array on areas not covered by a biological sample.
A method for colocalization obtains a first image encoding spatial abundance data for a nucleic acid from a biological sample through pixel values of subset of pixels in the first image. A second image is obtained encoding spatial abundance data for a non-nucleic acid analyte from the sample through pixel intensity values of subset of pixels in the second image. Thresholds of the first and second image are obtained. A determination, using a registration between the first and second images, of a first summation of the value of each pixel in the subset of pixels of the first image in which the corresponding pixel in the second image exceeds the second threshold value is made. The first summation, normalized to a summation of each value of each pixel in the subset of pixels of the first image, provides a measure of colocalization between the nucleic acid and non-nucleic acid analyte.
G16B 15/00 - ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
G16B 25/10 - Gene or protein expression profiling; Expression-ratio estimation or normalisation
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.
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 relates to methods, compositions, and kits for determining the location of target RNA in a biological sample and/or rescuing target RNA, from which spatial gene expression analysis is difficult to determine, from fresh-frozen biological samples.
C07K 16/28 - Immunoglobulins, e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
C12Q 1/00 - Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
G01N 33/566 - Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagent
12.
METHODS AND SYSTEMS FOR CHARACTERIZING ANTIGEN-BINDING MOLECULES EXPRESSED BY IMMUNE CELLS
The present disclosure relates generally to methods and systems for the characterization of antigen-binding molecules, e.g., B cell receptors, antibodies, and antigen-binding fragments thereof, using single-cell immune profiling methodologies. The methods and systems described herein permit rapid, high-throughput identification and characterization of antigen binding molecules having desired properties.
The present disclosure relates generally to compositions, methods, and systems for the characterization of binding charactersitics of antigen binding molecules, e.g., antibodies, using single-cell immune profiling methodologies. The compositions, methods and systems described herein permit rapid, high-throughput identification and characterization of antigen binding molecules having desired properties.
C07K 16/00 - Immunoglobulins, e.g. monoclonal or polyclonal antibodies
C07K 16/30 - Immunoglobulins, e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
15.
METHODS FOR PREPARING AND USING MHC MULTIMER REAGENTS COMPOSITIONS
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 using an oligonucleotide immobilized on the substrate via its 5' terminus. The method may comprise using a primer sequence for extension and/or a splint for ligation.
C12Q 1/6837 - Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
C40B 50/14 - 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
C40B 50/18 - 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 using a particular method of attachment to the solid support
18.
COMPOSITIONS AND METHODS FOR GENERATING MOLECULAR ARRAYS USING OLIGONUCLEOTIDE PRINTING AND PHOTOLITHOGRAPHY
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.
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 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.
in situin situin 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.
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.
Provided in some aspects are methods for light-controlled ?>? situ surface patterning of a substrate. Compositions such as nucleic acid arrays produced by the methods are also disclosed. In some embodiments, a method disclosed herein comprises using photocontrollable ligation of nucleic acid molecules with splints, wherein at least one of the splints comprises a photo-crosslinkable moiety. Photo-induced crosslinking of the splint to oligonucleotide molecules immobilized on a. substrate allows the splint to remain attached to the oligonucleotide molecules during subsequent rounds of photoresist application and removal. A large diversity of barcodes can be created in molecules on the substrate via sequential rounds of light exposure, hybridization, and ligation.
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.
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.
Provided herein are methods, kits, systems, and compositions for spatial analysis using one or more substrates (e.g., slides), wherein one of the substrates can have a spatial array. The methods disclosed herein include arrays having releasable capture probes that can interact with analytes or analyte derivatives and provide abundance and/or location of an analyte in a biological sample.
The present disclosure relates generally to compositions, methods, and systems for the characterization of antigen-binding molecules, e.g., multispecific T cell receptors, B cell receptors, antibodies, and antigen-binding fragments thereof, using single-cell immune profiling methodologies. The compositions, methods and systems described herein permit rapid, high-throughput identification and characterization of multispecific antigen binding molecules having desired properties.
The present disclosure relates generally to compositions and methods for identifying antibodies against membrane-bound antigens. More particularly, provided herein are compositions, methods, kits and systems for high throughput antibody discovery using lipoparticle-based antigens and barcoding methods.
A method for assessing the antigen binding specificity of an antigen binding molecule (ABM) expressed by one or more immune cells may include determining a first measurement of a target antigen bound to the antigen binding molecule and a second measurement of a control antigen bound to the antigen binding molecule. A specificity metric indicative of a likelihood of the antigen binding molecule binding to the target antigen at above a threshold may be determined based on the first measurement and the second measurement. The antigen binding molecule may be identified as exhibiting specificity towards the target antigen based on its specificity metric. Upon being identified as exhibiting specificity towards the target antigen, the antigen binding molecule may be further selected for having certain properties such as the presence of certain gene segments. Related systems and computer program products are also provided.
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.
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.
Provided herein are technologies for reducing fluid convection during processing and analysis of a biological sample. The provided technologies can include the use of a porous insert to limit fluid convection adjacent to a biological sample. In particular, provided technologies can include providing a substrate comprising a capture area, a biological sample comprising a cell disposed on the capture area, a buffer disposed on the biological sample, and a gasket disposed on the substrate, wherein the gasket provides a chamber comprising the lateral dimension of the capture area and includes a height above the capture area and biological sample; and a porous insert positioned in the chamber and in contact with the buffer and/or the biological sample, wherein the porous insert limits free flow space in the chamber, thereby reducing fluid convection adjacent to the biological sample.
A mold for processing a tissue sample includes a receptacle having a surface surrounded by at least one sidewall to define an interior space for receiving the tissue sample, and one or more guides extending from the surface in a direction perpendicular to a plane of the surface. The one or more guides are positioned within the receptacle to designate areas for receiving a portion of the tissue sample within the interior space, and the plurality of areas each comprise a predetermined area of the surface. A tissue specimen slide can be prepared by positioning one or more tissue samples in the designated areas on the surface of the mold, filling the mold with an embedding material to embed the tissue samples in the embedding material, cooling the embedding material in the mold to produce a tissue-embedded block, and removing the tissue-embedded block from the mold after cooling.
The present disclosure relates generally to compositions, methods, and systems for the characterization of T cell receptors and other antigen-binding molecules having T cell-like receptors, e.g., T cell receptor-like antibodies, using single-cell immune profiling methodologies. The compositions, methods and systems described herein permit rapid, high-throughput identification and characterization of T cell, and/or T cell-like, receptors having desired properties.
G01N 33/50 - Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
C12Q 1/6804 - Nucleic acid analysis using immunogens
G01N 33/542 - Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
C07K 14/74 - Major histocompatibility complex (MHC)
G01N 33/58 - Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
G01N 33/68 - Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
36.
COMPOSITIONS AND METHODS FOR CHARACTERIZING ANTIGEN BINDING MOLECULES FROM SINGLE CELLS
The present disclosure relates generally to compositions, methods, and systems for the characterization of antigen binding molecules, e.g., antibodies, using single-cell immune profiling methodologies. The compositions, methods and systems described herein permit rapid, high-throughput identification and characterization of antigen binding molecules having desired properties.
The present disclosure relates generally to compositions comprising modified MHC class I and MHC class II molecules. Methods and systems for the characterization of T cell receptors and other antigen-binding molecules having T cell-like receptors, e.g., T cell receptor-like antibodies, using single-cell immune profiling methodologies are also disclosed. The compositions, methods and systems described herein permit rapid, high-throughput identification and characterization of T cell, and/or T cell-like, receptors having desired properties.
Disclosed herein are modified split mirrored transposons or double-stranded split mirrored transposon (SMT) compositions and methods of using SMTs. The methods include producing SMTs and using SMTs for nucleic acid analysis.
in situin 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.
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 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.
Provided herein are methods of detecting a target nucleic acid of interest for spatial gene expression analysis in a sample using barcoded templated ligation probes.
Systems and methods for evaluating a biological sample on a substrate are provided. An image of the biological sample and glyphs on the substrate are displayed on a display as a plurality of pixels. Respective indications are received of coordinates within the image of the glyph locations. These and a reference fiducial pattern that includes the plurality of glyphs are used to calculate and display an initial alignment between the image and the fiducial pattern. The alignment is updated through manual user adjustments to glyph coordinates. A set of pixels in the plurality of pixels depicting the biological sample are received from a user. Identification of each capture spot in a plurality of capture spots encompassed by the set of pixels is outputted to an output file, with each respective capture spot being identified within the image for the output file based on the updated alignment.
A61K 39/00 - Medicinal preparations containing antigens or antibodies
C07K 16/28 - Immunoglobulins, e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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 33/574 - Immunoassay; Biospecific binding assay; Materials therefor for cancer
Provided herein in some aspects are methods, compositions, kits, and systems for performing multiplexed single-cell analysis on an in situ platform, providing alternatives which have a higher cell throughput and/or lower cost per cell compared to current single-cell analysis techniques. In some embodiments, the methods disclosed herein comprise using labeling agents that comprise sample- specific barcodes and/or cell feature specific barcodes (e.g., analyte specific barcodes) to label single-cell populations, immobilizing the labeled cells, and performing in situ detection of the labeling agents and/or other features including cellular analytes of the labeled cells.
Techniques for spectral unmixing and decoding for in situ analysis are provided. Input hypercube data comprising voxel data, raw channel data, and decoding round data are obtained, and an initial iteration of codeword data determination is performed. Performing the initial iteration includes generating initial feature data and initial unmixed fluorescence data, generating initial uncorrected codeword data, and generating initial corrected codeword data. One or more subsequent iterations of codeword data determination are then performed based on the input hypercube data and based on feature data, unmixed fluorescence data, uncorrected codeword data, and corrected codeword data from one or more previous iterations. When it is determined that one or more convergence conditions have been satisfied, output comprising an optimized estimate of fluorescence data, and an optimized estimate of uncorrected codeword data, and an optimized estimate of corrected codeword data is generated.
The present disclosure relates generally to compositions comprising antigen-binding molecules that bind to a severe acute respiratory syndrome coronavirus (SARS-CoV), and/or comprising nucleic acids that encode such antigen-binding molecules, as well as therapeutic and diagnostic methods for using such compositions.
A method of capturing analytes from a sample is provided. A first substrate including a sample is mounted on a first member of a sample handling device and mounting a second substrate including an array of capture probes on a second member of the sample handling device. A first reagent medium is applied to the first and/or second substrate and moving the first member and/or the second member to fluidically couple the sample and the array of capture probes via the first reagent medium. The sample and the array of capture probes are fluidically decoupled by moving the first member and/or the second member and a second reagent medium is applied to the first and/or second substrate and moving the first member and/or the second member to fluidically couple the sample and the array of capture probes via the second reagent medium. Related systems and apparatuses are also provided.
Methods and systems for performing in situ decoding are described that minimize optical crowding, thereby improving decoding accuracy. The methods may comprise, e.g., receiving images of a biological sample acquired during a cyclical decoding process; detecting a series of detectable signals (ON signals) or absence thereof (OFF signals) at one or more locations in the biological sample corresponding to one or more barcoded target analytes; determining a code word based on the series of ON and OFF signals that corresponds to a barcode for each of the one or more barcoded target analytes, where the one or more code words are assigned to the one or more barcoded target analytes based on a minimax decision rule to minimize a density of ON signals detected in the images of the series of images; and identifying the one or more barcoded target analytes based on the one or more determined code words.
Devices, systems, and their methods of use for droplet generation are provided. The devices, systems, and their methods have a droplet source region that is coated with a fluoropolymer silane coating agent that provides a surface coating having a total fluorine weight percent of at least 57%.
C08G 65/00 - Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
C08G 65/336 - Polymers modified by chemical after-treatment with organic compounds containing silicon
C09D 171/00 - Coating compositions based on polyethers obtained by reactions forming an ether link in the main chain; Coating compositions based on derivatives of such polymers
B01F 25/314 - Injector mixers in conduits or tubes through which the main component flows wherein additional components are introduced at the circumference of the conduit
B01F 33/3011 - Micromixers using specific means for arranging the streams to be mixed, e.g. channel geometries or dispositions using a sheathing stream of a fluid surrounding a central stream of a different fluid, e.g. for reducing the cross-section of the central stream or to produce droplets from the central stream
53.
SYSTEMS AND METHODS FOR SPATIAL ANALYSIS OF ANALYTES USING FIDUCIAL ALIGNMENT
Systems and methods for spatial analysis of analytes are provided. A data structure is obtained comprising an image, as an array of pixel values, of a sample on a substrate having intersecting border regions, fiducial markers encoding N-digit codes, and a set of capture spots, where at least two border regions includes a fiducial marker. The pixel values are analyzed to identify locations of fiducial markers. The locations are aligned with locations of reference fiducial markers in a template using an alignment algorithm to obtain a final transformation between the fiducial markers in the image and the reference fiducial markers in the template. The final transformation and a coordinate system of the template are used to register the image to the set of capture spots. The registered image is then analyzed in conjunction with spatial analyte data associated with each capture spot, thereby performing spatial analysis of analytes.
Provided herein are methods of analyzing an analyte in a fixed biological sample on a first substrate that has been stored for a long period of time, the method comprising: (a) hybridizing a first probe and a second probe to the analyte of the fixed biological sample; (b) coupling the first probe and the second probe, thereby generating a connected probe; (c) aligning the first substrate with a second substrate comprising an array, wherein the array comprises 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; (d) when the biological sample is aligned with at least a portion of the array, (i) releasing the connected probe from the analyte and (ii) migrating the connected probe from the biological sample to the array; and (e) hybridizing the connected probe to the capture domain.
Provided herein are methods, compositions, and systems for identifying spatial expression of analytes is an archived fixed biological tissue sample that had been stored for an extended period of time at sub-optimal storage conditions.
In some aspects disclosed herein are methods and compositions for detecting analytes such as a plurality of nucleic acid molecules in a biological sample, said method comprising generating and analyzing a first detectable signal and a second detectable signal corresponding to a first target sequence and a second target sequence, respectively. The first and second target sequences can be present and/or expressed at different levels in the biological sample.
Disclosed are assemblies, systems, and methods for transilluminating a sample disposed within a device. An assembly includes a first optically transparent substrate configured to receive a biological sample, a second optically transparent substrate having a top surface, a bottom surface, and a plurality of sides. The first substrate contacts the top surface of the second substrate. The assembly includes at least one light source configured to illuminate at least one of the plurality of sides of the second substrate and a light scattering layer on the bottom surface of the second substrate. The light scattering layer is configured to scatter light from the light source. The assembly includes a thermal control module coupled to the second substrate and configured to control the temperature of the second substrate.
B01L 3/00 - Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
F21V 8/00 - Use of light guides, e.g. fibre optic devices, in lighting devices or systems
G01N 21/77 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
The present disclosure in some aspects relates to methods and compositions for detecting and quantifying multiple analytes present in a biological sample by rolling circle amplification (RCA). In some aspects, the methods and compositions provided herein address issues associated with control of signal spot size and intensity for RCA products (RCPs) in a sample. In some aspects, provided herein are methods and compositions (e.g., probes and/or reaction mixtures) for slowing down RCA and/or decreasing the size of RCA products. In some embodiments, nucleotides or nucleotide analogs having a hydrophobic group (e.g., a hydrophobic modification on the base) are included in nucleotides for RCA and incorporated into the RCP product, leading to reduction of RCP size without the need of crosslinking an incorporated hydrophobic nucleotide or nucleotide analog to the RCP itself or another molecule.
A method of self-testing an imaging system of a sample handling apparatus is provided. Systems and non-transitory computer readable mediums performing the method are also provided.
G06T 7/33 - Determination of transform parameters for the alignment of images, i.e. image registration using feature-based methods
G06T 7/80 - Analysis of captured images to determine intrinsic or extrinsic camera parameters, i.e. camera calibration
G01B 21/04 - Measuring arrangements or details thereof, where the measuring technique is not covered by the other groups of this subclass, unspecified or not relevant for measuring length, width, or thickness by measuring coordinates of points
G02B 21/34 - Microscope slides, e.g. mounting specimens on microscope slides
61.
METHODS FOR IMPROVING SENSITIVITY OF IMMUNE PROFILING USING OLIGO-TAGGED ANTIGENS
Provided herein, inter alia, are methods and compositions for inhibiting DNase II- mediated degradation of reporter nucleic acid molecules coupled to antigens of interest, and thus improving sensitivity of single-cell immune profiling workflows.
The disclosure provides recombinant reverse transcriptases comprising one or more DNA binding domains conjugated to an engineered reversed transcriptase that have been modified to exhibit one or more altered reverse transcriptase related activities such as but not limited to altered template switching efficiency, altered transcription efficiency or both. The disclosure further provides compositions and kits comprising the recombinant reverse transcriptase enzymes and methods of producing, amplifying or sequencing nucleic acid molecules using these fusion reverse transcriptase enzymes.
Systems and methods are provided for identifying immune receptor sequences including receiving known immune receptor sequences, generating a trained multi-layer model for immune receptor discovery, generating a set of new immune receptor sequences, and applying the trained multi-layer model to the set of new immune receptor sequences to identify an immune receptor sequence based on an associated second fitness score for each new immune receptor sequence. In certain embodiments of the method, each sequence has an associated first fitness score which includes training a first layer of the multi-layer model using a collection of known proteins, tuning the first layer to produce a second layer using a collection of known immune receptor sequences, and training a third layer using the set of known immune receptor sequences, each sequence including the associated first fitness score.
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
Methods and systems for performing multi-resolution in situ decoding are described. The method may comprise, for example, acquiring at least one first image of a biological sample at a first optical resolution; identifying locations for a plurality of target analytes based on the at least one first image; acquiring at least one second image of the biological sample at a second optical resolution in at least one decoding cycle of a plurality of decoding cycles used for in situ decoding of the target analytes; and extracting signal intensity data for signals associated with all or a portion of the target analytes from the at least one second image based on the locations for the target analytes identified in the at least one first image. In some instances, the method may further comprise using the signal intensity data extracted from the at least one second image to decode the target analytes.
in situin situ detection of analytes and spatial analysis using, e.g., a sequencing readout. The biological sample, e.g. a tissue sample, is placed on a first substrate, the method comprising: (a) contacting the biological sample with one or more nucleic acid probes that directly or indirectly hybridize to a set of first analytes or complements or amplification products thereof in the biological sample; (b) detecting in the biological sample the one or more nucleic acid probes at a spatial location of the biological sample; (c) hybridizing a first probe and a second probe to a second analyte, wherein the first probe comprises a sequence that is substantially complementary to a first sequence of the second analyte, the second probe comprises a sequence that is substantially complementary to a second sequence of the second analyte, and wherein the second probe comprises a capture probe binding domain; (d) coupling the first probe and the second probe, thereby generating a connected probe; (e) mounting the first substrate on a first member of a support device, the first member configured to retain the first substrate; (f) mounting a second substrate comprising an array on a second member of the support device, the second member configured to retain the second substrate, the 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; (g) applying a reagent medium to the first substrate and/or the second substrate; (h) operating an alignment mechanism of the support device to move the first member and/or the second member such that at least a portion of the biological sample is aligned with at least a portion of the array and the portion of the biological sample and the portion of the array contact the reagent medium, and such that the first substrate and the second substrate are separated by a distance of less than 50 micrometers; (i) when the biological sample is aligned with at least a portion of the array, (i) releasing the connected probe from the second analyte and (ii) migrating the connected probe from the biological sample to the array; and (j) hybridizing the connected probe to the capture domain. In other embodiments a capture probe with a capture handle is used in stead of the ligation probes. In still other embodiments the capture probe with capture handle is used as probes in steps a) and b). In still other embodiments an in-situ analysis is carried out in stead of steps a) and b).
Systems and methods for identifying regions of aneuploidy in a tissue include obtaining nucleic acid sequence reads, each including a spatial barcode, associating the read with a feature in a two-dimensional array of features on a substrate contacting the tissue, and a unique molecular identifier (UMI). The reads serve to determine a count data structure comprising, for each of a plurality of genomic regions, a respective UMI count for each feature in the two-dimensional array of features on the substrate. For each feature in the array of features, a respective bin count is made for each respective bin in a plurality of bins corresponding to the respective feature, where the plurality of bins span a genome. Copy number state respective features in the array are determined using feature bin counts. The copy number state of each feature in the array of features serves to identify regions of tissue aneuploidy.
Provided herein are methods for identification of antigen binding molecules such as antibodies from a sample by exposing the antigen binding molecules to an antigen conjugated to an oligonucleotide.
G01N 33/537 - Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
Provided herein are methods for spatial analysis that captures non-poly(A)-containing RNA molecules, such as long non-coding RNAs and microRNAs. Methods, kits, and compositions for spatial analysis using targeted RNA capture using randomer capture probes are disclosed herein.
Systems and methods for evaluating one or more biological samples are provided. A dataset is obtained from nucleic acid sequencing of the biological samples. The dataset comprises a discrete attribute value for each of a plurality of reference sequences for each entity in a plurality of entities in the biological samples. A two-dimensional spatial arrangement of the plurality of entities is indexed, each entity independently assigned a unique two-dimensional position in a k-dimensional binary search tree, and the spatial arrangement is displayed. A user selection of a subset of the displayed arrangement is received. Each entity that is a member of the subset is determined using the k-dimensional binary search tree, thus identifying a subset of entities. Each entity in the subset of entities is assigned to a user-provided category, and the dataset is modified to store an association of each entity in the subset to the category.
G16B 45/00 - ICT specially adapted for bioinformatics-related data visualisation, e.g. displaying of maps or networks
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
Provided herein are methods for identification chimeric antigen receptors in immune cells as well as method for identification and characterization of immune cells expressing those chimeric antigen receptors.
Systems and methods for overlaying image data for a biological sample on spatial analyte data are provided. A first image of the sample on a first substrate and a second image of the sample on the first substrate overlayed on a second substrate are obtained. The second substrate includes spatial fiducials and capture spots. At least one of the first substrate and the second substrate is transparent. A registration for the first image and the second image is determined, using a first pattern of the sample in the first image and a second pattern of the sample in the second image. The registration is used to overlay the first image onto a spatial dataset including spatial analyte data for the capture spots from the sample. A frame of reference of the spatial dataset is known with respect to the second image, based on the spatial fiducials of the second image.
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.
Provided herein are reaction mixtures, compositions, systems, methods, and kits for assessing enzymatic activity. Aspects of the disclosure include use of a cleavable substrate comprising a first portion linked to a second portion designed to be the target of an enzyme. A substrate barcoded oligonucleotide (SBO) construct may be linked to the first portion and a blocking construct may be linked to the second portion. A rate of substrate cleavage may be measured to determine an enzymatic activity of a given enzyme.
In some aspects, the present disclosure relates to methods for reducing the detection of false positive ligation events. In some aspects, the method comprises use of a double split (or "split split") probe. The methods herein have particular applicability in reducing the detection of false positive ligation events when using ligases that have high ligation efficiency but low specificity (e.g., SplintR® ligase). Also provided are kits comprising probes for use in such methods.
Provided herein are methods, compositions, and kits including single-chain antibody nucleic acid libraries and methods of displaying single-chain antibodies and screening single-chain antibodies on a spatial array.
The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying multiple analytes present in a biological sample. In some aspects, the methods and compositions provided herein address one or more issues associated with the stability and/or size of nucleic acid structures, such as RCPs, in the biological sample without the use of exogenously added oligonucleotide compaction probes. In some embodiments, provided herein are methods involving the use of self-hybridizing hybridizing regions for compacting and/or stabilizing nucleic acid concatemers (e.g., RCPs). In some embodiments, dynamic inter-strand annealing between tandem units of an RCP is used for compaction and/or stabilization. In some embodiments, short palindromic regions in an RCP are used for compaction and/or stabilization.
The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying multiple analytes present in a biological sample. In some aspects, the methods and compositions provided herein address issues associated with the heterogeneity of analyte abundance (e.g., gene expression levels) and variations among reactions at different locations of a sample (e.g., amplification reaction starting earlier at one location than another location). In some aspects, a method disclosed herein provides a tighter distribution of signal spot size and intensity in a sample, as compared to methods that result in a wide and heterogeneous size and intensity distribution of signal spots.
The present disclosure provides methods of processing or analyzing a sample. A method for processing a sample can comprise barcoding a nucleic acid molecule, circularizing the resultant product, and performing an extension reaction, without the need of a template switching molecule. One or more processes of the methods described herein can be performed within a partition, such as a droplet or well.
Devices, kits, and their methods of use for lysing and/or purifying biological particles, e.g., nuclei are provided. One or more thixotropic layers can be employed in a vessel to purify biological particles. A device with sharp features may be employed to lyse biological particles or the contents thereof.
Provided herein are methods for selective silencing of one or more probes used in templated ligation, or RNA-templated ligation. In some embodiments, the selective silencing is achieved using one or more blocker oligonucleotides.
In some aspects, the present disclosure relates to methods for reducing the crowding of signals, for example optical crowding, that can occur when nucleic acids are detected in a sample in multiplex, which can make it difficult to resolve individual signals and can lead to a reduced dynamic range. In some aspects, the present disclosure relates to methods for reducing signal crowding in the detection of multiple target nucleic acid sequences in a sample, e.g., using hybridization probes, wherein signal crowding from said hybridization probes is reduced. The methods herein have particular applicability in the detection of barcode sequences by sequencing-by-hybridization (SBH) methods, including those relying on combinatorial labelling schemes and decoding of the barcodes by sequential cycles of decoding using hybridization probes. Also provided are kits comprising probes for use in such methods.
The present disclosure provides methods for biological sample preparation and analysis that use a chopping and fixing treatment in the preparation of dissociated fixed cells from biological tissue for use in either bulk assays, or in single-cell/single-nucleus assays, such as partition-based assays. The disclosure also provides assay methods, including partition-based methods, for use of the fixed cells prepared from biological tissue that can optionally be used in combination with un-fixing treatments. Kits comprising dissociation reagents, un-fixing agents, and other assay reagents for use in the methods are also provided.
The disclosure provides engineered reverse transcriptase enzymes that have been modified to enhance their enzymatic to increase their processivity, template switching efficiency, binding affinity, and/or transcription efficiency and to decrease their RNAse H activity. The disclosure further provides compositions and kits comprising the engineered reverse transcriptase enzymes and methods of producing, amplifying or sequencing nucleic acid molecules using these reverse transcriptase enzymes.
Provided herein are methods for validating a unique molecular identifier (UMI) of a barcoded nucleic acid molecule selected from a complex pool of barcoded nucleic acid molecules. The barcoded nucleic acid molecule may be a nucleic acid sequence of interest. As such, upon validating the unique molecular identifier, the barcoded nucleic acid molecule may be processed for further analysis, enrichment, and/or cloning. For example, the barcoded nucleic acid molecule may be enriched using primers that target the identification sequences including the validated unique molecular identifier. The barcoded nucleic acid molecule may be subsequently cloned, and the protein products can be analyzed.
The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying analytes present at high levels, such as highly expressed genes in a sample. In some embodiments, a probe-resolution barcode sequence disclosed herein does not specifically correspond to any particular target analyte(s) but can be used to resolve dense optical signals due to spatially overlapping signals associated with different molecules of a target analyte, thereby enabling resolution of signals in a dense "spot" and accurate counting of spots associated with molecules that are in spatial proximity. Also provided are kits comprising probes for use in such methods.
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12Q 1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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
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
C40B 40/02 - Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
C40B 50/06 - Biochemical methods, e.g. using enzymes or whole viable microorganisms
C40B 40/10 - Libraries containing peptides or polypeptides, or derivatives thereof
C40B 60/04 - Integrated apparatus specially adapted for both screening libraries and identifying library members
C07K 16/00 - Immunoglobulins, e.g. monoclonal or polyclonal antibodies
91.
SAMPLE ANALYSIS USING ASYMMETRIC CIRCULARIZABLE PROBES
The present disclosure in some aspects relate to a direct RNA (dRNA) detection approach incorporating the use of circularizable probes (e.g., padlock probes) having asymmetric arms, rolling circle amplification of the ligated circularizable probes, and in situ detection (e.g., using hybridization-based in situ sequencing) for multiplexed spatial analysis of nucleic acid sequences (e.g., short sequences such as SNPs and point mutations) in a biological sample. In some aspects, compositions and methods disclosed herein improve detection specificity, reduce false positive signal detection, and/or maintain or improve detection efficiency.
Provided herein are methods of enhancing spatial resolution of an analyte using sandwich maker system. The methods and systems used herein include a first substrate that includes a plurality of probes that include a capture domain and a spatial domain and a second substrate that includes a plurality of probes comprising a capture domain and a spatial domain.
Provided herein are methods for capturing an analyte from a first region of interest of a biological sample on a substrate, where the biological sample comprises the first region of interest and a second region, and where the method includes contacting the second region with a sealant in order to create a hydrophobic seal thereby preventing an interaction between an analyte from the second region with a capture domain of a capture probe.
The application provides compositions including engineered fusion reverse transcriptases with at least one altered reverse-transcriptase related activity. The engineered fusion reverse transcriptases or reverse transcription enzymes unexpectedly exhibit one or more altered reverse transcriptase related activities such as but not limited to altered template switching efficiency, altered transcription efficiency or both.
Pressure control systems and methods are provided that aid in the control of fluidic or pneumatic devices, by improving the ability to control pressure independently and simultaneously on multiple channels, which in turn permits pressure changes on the channels to occur more quickly and more precisely. In order to match rise/fall times between steps on different channels that may be of different magnitudes, various embodiments slow down fast steps such that they match the "default" rate of slower steps, such as by using a step partitioning method or breaking a single step into substeps with a pause inserted between substeps of necessary duration such that the complete step time matches the target step time. The provided systems and methods may utilize a combination of proportional-integral-derivative (PID) control loop and discrete pressure steps to achieve faster, more accurate control over pressure rises and pressure falls.
Provided herein are methods of testing a biological sample for efficacy of detection of a target nucleic acid where the method includes RNA templated ligation including generating a sequence that is complementary to the hybridized ligation product that includes labelled nucleotides and detecting a signal corresponding to the ligation product on the substrate, thereby determining the efficacy of detection of the target nucleic acid.
Provided herein are methods for determining the mislocalization of an analyte by capturing the analyte on an array and measuring the mislocalization distance.
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.
A recombinant ligase composition including a recombinantly-produced ligase isolated from Acanthocystis turfacea chlorella virus 1 (ATCV-1) fused to at least one polynucleotidebinding polypeptide is provided. Compositions containing the recombinant ligase composition, vectors encoding the recombinant ligase composition, and methods of making and using the recombinant ligase composition are also provided.
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.
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