The disclosure provides systems and methods for droplet processing. For example, a method can include providing a first droplet and a second droplet. In some cases, the first droplet may have a first concentration of a reagent and the second droplet may have a second concentration of the reagent. The second droplet may comprise a bead or a biological particle. The method can also include subjecting the first droplet and the second droplet to conditions sufficient to transfer the reagent from the first droplet to the second droplet, thereby decreasing the first concentration in the first droplet and increasing the second concentration in the second droplet.
Provided herein are methods for image segmentation. An image of a sample having a plurality of nuclei is received. The image comprises a plurality of pixels arranged in a first dimension and a second dimension. The plurality of pixels indicates a signal from a nuclear stain of the plurality of nuclei. For each pixel of the plurality of pixels, a pixel classification is determined, thereby generating a pixel classification map corresponding to the image. Determining the pixel classification comprises determining a first classification of the pixel corresponding to the first dimension and determining a second classification of the pixel corresponding to the second dimension. A segmentation mask is determined based on the pixel classification map.
G06V 20/69 - Objets microscopiques, p. ex. cellules biologiques ou pièces cellulaires
G06V 10/26 - Segmentation de formes dans le champ d’imageDécoupage ou fusion d’éléments d’image visant à établir la région de motif, p. ex. techniques de regroupementDétection d’occlusion
G06V 10/764 - Dispositions pour la reconnaissance ou la compréhension d’images ou de vidéos utilisant la reconnaissance de formes ou l’apprentissage automatique utilisant la classification, p. ex. des objets vidéo
G06V 10/774 - Génération d'ensembles de motifs de formationTraitement des caractéristiques d’images ou de vidéos dans les espaces de caractéristiquesDispositions pour la reconnaissance ou la compréhension d’images ou de vidéos utilisant la reconnaissance de formes ou l’apprentissage automatique utilisant l’intégration et la réduction de données, p. ex. analyse en composantes principales [PCA] ou analyse en composantes indépendantes [ ICA] ou cartes auto-organisatrices [SOM]Séparation aveugle de source méthodes de Bootstrap, p. ex. "bagging” ou “boosting”
3.
ELECTROPHORETIC SYSTEM AND METHOD FOR ANALYTE CAPTURE
An electrophoretic system is provided for analyte capture from a biological sample. The electrophoretic system can be used to permeabilize the sample to allow analytes to be released from the sample. For example, the sample can be contacted with capture probes attached to a substrate, and an electric field created by the electrophoretic system can cause analytes to be released from the cell, and effectively migrate toward and bind to the capture probes attached to the substrate.
The present disclosure relates in some aspects to methods and compositions for in situ analysis involving catalytic de-crosslinking of biological samples.
C12Q 1/68 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir des acides nucléiques
C12Q 1/6806 - Préparation d’acides nucléiques pour analyse, p. ex. pour test de réaction en chaîne par polymérase [PCR]
C12Q 1/6816 - Tests d’hybridation caractérisés par les moyens de détection
A method for determining sample detachment from a substrate is provided. A plurality of images of a sample on a substrate are received where the plurality of images includes a plurality of fields of view of the sample. At least one focus score is determined for each image in the plurality of images. Based on the determined focus scores, one or more portions of the sample are determined as being detached from the substrate.
G06V 10/50 - Extraction de caractéristiques d’images ou de vidéos en effectuant des opérations dans des blocs d’imagesExtraction de caractéristiques d’images ou de vidéos en utilisant des histogrammes, p. ex. l’histogramme de gradient orienté [HoG]Extraction de caractéristiques d’images ou de vidéos en utilisant l’addition des valeurs d’intensité d’imageAnalyse de projection
Provided are methods, systems, and kits for circularization-based dual 3′/5′ assays for sequence analysis of barcoded nucleic acids. The circularization-based dual 3′/5′ assays included single cell sequencing assays and spatial sequencing assays.
Methods and kits for sample preparation are provided, wherein a sample comprising at least one cell or tissue is contacted sequentially with a hydrophobic crosslinker, and then a hydrophilic crosslinker. The method prevents cell and tissue analytes from diffusing out of the at least one cell or tissue, such that the cell and tissue analytes can be analyzed and/or imaged.
C12N 5/00 - Cellules non différenciées humaines, animales ou végétales, p. ex. lignées cellulairesTissusLeur culture ou conservationMilieux de culture à cet effet
The present disclosure relates to compositions and methods for generating capture probes on a substrate for identifying the location of analytes in a biological sample.
C12Q 1/6874 - Méthodes de séquençage faisant intervenir des réseaux d’acides nucléiques, p. ex. séquençage par hybridation [SBH]
C12Q 1/6876 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes
C12Q 1/6881 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour le typage de tissu ou de cellule, p. ex. sondes d’antigène leucocytaire humain [HLA]
G01N 33/483 - Analyse physique de matériau biologique
G01N 35/00 - Analyse automatique non limitée à des procédés ou à des matériaux spécifiés dans un seul des groupes Manipulation de matériaux à cet effet
G02B 21/16 - Microscopes adaptés pour éclairage ultraviolet
G02B 21/26 - PlatinesMoyens de réglage pour celles-ci
G02B 21/34 - Lames de microscope, p. ex. montage d'échantillons sur des lames de microscope
G02B 21/36 - Microscopes aménagés pour la photographie ou la projection
10.
NUCLEIC ACID SEQUENCING METHODS WITH CONTROL SEQUENCES
The present disclosure relates in some aspects to methods, systems, and kits for nucleic acid sequencing using a control sequencing primer to perform a plurality of cycles of a nucleic acid sequencing reaction of a control sequence comprising a known sequence. In some cases, the control sequencing primer is detected to identify the location of a plurality of copies of the control sequence. In certain embodiments, for a cycle of the plurality of cycles at the identified location, the method comprises comparing a signal from the nucleic acid sequencing reaction to an expected signal from the known sequence, thereby determining noise in the signal and/or identifying an error in the nucleic acid sequencing reaction.
Provided herein are systems and methods for analyzing biomolecules (e.g., nucleic acid molecules, proteins). A method of analyzing a target ribonucleic acid (RNA), can include: (a) providing: (i) a target RNA, comprising a first target sequence and a second target sequence, and (ii) at least one probe including a first probe end hybridized to the first target sequence, and a second probe end hybridized to the second target sequence, wherein the first probe end hybridized to the first target sequence and the second probe end hybridized to the second target sequence are separated by a gap region. The method can further include filling the gap region and generating a probe-linked nucleic acid molecule including the at least one probe and the gap region.
The present disclosure relates in some aspects to methods and compositions for sequencing nucleic acids in a 3D hydrogel. In some aspects, the methods comprise generating 3D hydrogels comprising immobilized RCPs that are generated from isolated nucleic acids, and sequencing the RCPs to determine the sequences of the isolated nucleic acids. In some embodiments, the isolated nucleic acids comprise sequencing libraries.
The present disclosure relates to methods for analyzing target nucleic acids in a biological sample using Argonaute proteins and single cell RNA-templated ligation probe chemistry. Variant sequences (e.g., single nucleotide variations such as SNPs or point mutations) in a plurality of target nucleic acids in a cell or tissue sample are analyzed in the sample. The application is drawn to assay methods, and further drawn to compositions and kits for use in accordance with the methods. One or more processes of the methods described herein may be performed within a partition, such as a droplet or well.
An imaging system, comprising: a tube lens; a first image sensor; a second image sensor; an objective disposed to direct emission light from a focal plane of the objective to the tube lens. The first image sensor and the second image sensor are arranged at focal planes of the tube lens. A beamsplitter is disposed along a first optical axis between the tube lens and the first image sensor to intercept the path of the emission light. The beamsplitter comprises: an ingress face arranged perpendicular to the first optical axis, a transmission-reflection face arranged oblique to the ingress face and downstream of the ingress face along the first optical axis, wherein the transmission-reflection face is arrange to transmit a first component of the emission light along the first optical axis and reflect a second component of the emission light along a second optical axis, a first egress face arranged downstream of the transmission-reflectance face along the first optical axis, and a second egress face arranged downstream of the transmission reflectance face along the second optical axis. The first egress face is arranged perpendicular to the first optical axis, and the second egress face is arranged perpendicular to the second optical axis.
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.
G06T 7/168 - DécoupageDétection de bords impliquant des procédés de transformation de domaine
G06T 7/33 - Détermination des paramètres de transformation pour l'alignement des images, c.-à-d. recalage des images utilisant des procédés basés sur les caractéristiques
Methods for determining a location of a feature on an array include: (a) providing a first array with a first plurality of features immobilized on a first substrate; (b) providing a second array with a second plurality of features immobilized on a second substrate; (c) aligning the first array with the second array; (d) hybridizing a first barcoded oligonucleotide of the first array to a second barcoded oligonucleotide of the second array, thereby producing a combined nucleic acid that includes first and second spatial barcodes; (e) determining all or a portion of the sequence of the combined nucleic acid; and (f) identifying the second barcoded oligonucleotide associated with the first barcoded oligonucleotide in the combined nucleic acid, and determining the location of a second feature in the second array.
C12Q 1/6874 - Méthodes de séquençage faisant intervenir des réseaux d’acides nucléiques, p. ex. séquençage par hybridation [SBH]
C12Q 1/6876 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes
C12Q 1/6881 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour le typage de tissu ou de cellule, p. ex. sondes d’antigène leucocytaire humain [HLA]
G01N 33/483 - Analyse physique de matériau biologique
G01N 35/00 - Analyse automatique non limitée à des procédés ou à des matériaux spécifiés dans un seul des groupes Manipulation de matériaux à cet effet
G02B 21/16 - Microscopes adaptés pour éclairage ultraviolet
G02B 21/26 - PlatinesMoyens de réglage pour celles-ci
G02B 21/34 - Lames de microscope, p. ex. montage d'échantillons sur des lames de microscope
G02B 21/36 - Microscopes aménagés pour la photographie ou la projection
17.
METHODS AND COMPOSITIONS FOR IN SITU ANALYSIS OF DNA METHYLATION
The present disclosure generally relates to methods and compositions for interrogating and/or analyzing DNA methylation in a biological sample. In some aspects, the present disclosure relates to methods for determining the methylation status of a region of interest of genomic DNA. In some aspects, the methylation status is analyzed by interrogating converted DNA in which the sequence of the converted DNA is indicative of the methylation state of the DNA. In some aspects, the methods comprise generating a collective signal that is based on the methylation states of a plurality of sequences or residues in the DNA, and that is representative of the methylation status of the region of interest as a whole.
C12Q 1/6874 - Méthodes de séquençage faisant intervenir des réseaux d’acides nucléiques, p. ex. séquençage par hybridation [SBH]
C12Q 1/6886 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour les maladies provoquées par des altérations du matériel génétique pour le cancer
18.
COMPOSITIONS AND METHODS FOR IMPROVED ON-TARGET SPATIAL PROFILING
Compositions and methods for enhanced resolution of spatial profiling of nucleic acids in biological samples including single-stranded (ss)DNA regions have been developed. The methods reduce or prevent off-target binding of analyte-binding probes with ssDNA within biological samples to enhance resolution, sensitivity, specificity and/or accuracy of spatial analysis of targeted analytes. The methods comprise extension using ssDNA as a template to provide a complementary second DNA strand, forming a "blocked sample" having less ssDNA than the biological sample prior to being treated in accordance with the disclosed methods. In some embodiments, the methods employ a multiplicity of block primers having designed or random sequences that hybridize with accessible regions of ssDNA within a biological sample. In some embodiments, the methods employ extension of one or more regions of ssDNA in a biological sample using a primer-free polymerase to provide a blocked sample.
This disclosure provides methods for spatial profiling of biological analytes present in a biological sample. Methods include generating feature arrays using a master/copy format using recessed arrays, and methods for using such arrays. For example spatially-tagged analyte capture analytes can be used in spatial detection in methods to determine the location of analytes (e.g., proteins) in biological samples.
C12Q 1/6874 - Méthodes de séquençage faisant intervenir des réseaux d’acides nucléiques, p. ex. séquençage par hybridation [SBH]
C12Q 1/6876 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes
C12Q 1/6881 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour le typage de tissu ou de cellule, p. ex. sondes d’antigène leucocytaire humain [HLA]
G01N 33/483 - Analyse physique de matériau biologique
G01N 35/00 - Analyse automatique non limitée à des procédés ou à des matériaux spécifiés dans un seul des groupes Manipulation de matériaux à cet effet
G02B 21/16 - Microscopes adaptés pour éclairage ultraviolet
G02B 21/26 - PlatinesMoyens de réglage pour celles-ci
G02B 21/34 - Lames de microscope, p. ex. montage d'échantillons sur des lames de microscope
G02B 21/36 - Microscopes aménagés pour la photographie ou la projection
20.
METHODS, COMPOSITIONS, AND KITS FOR MULTIPLE BARCODING AND/OR HIGH-DENSITY SPATIAL BARCODING
The present disclosure features methods, compositions, and kits for multiple barcoding, high-density barcoding, and/or selective release of barcoded capture probes to capture analytes, or proxies thereof, from a biological sample.
Provided herein are methods, compositions, and kits for the transposome-mediated capture of analytes on a substrate including spatially barcoded capture probes.
The present disclosure provides systems and methods for measuring one or more analytes at the single cell level. In some instances, sequencing of a composite barcode sequence may identify a type of analyte, identify a cell, and determine that the type of analyte originated from the cell. In some instances, a probability that a type of analyte originated from the cell may be determined. Multiple types of analytes may be identified to have originated from the cell, and/or the respective probabilities determined. An analyte may be a protein, such as a surface-bound protein or an internal protein. An analyte may be a metabolite or other small molecule. An analyte may be any constituent of a cell.
G01N 33/543 - Tests immunologiquesTests faisant intervenir la formation de liaisons biospécifiquesMatériaux à cet effet avec un support insoluble pour l'immobilisation de composés immunochimiques
B01L 3/00 - Récipients ou ustensiles pour laboratoires, p. ex. verrerie de laboratoireCompte-gouttes
C12Q 1/6804 - Analyse d’acides nucléiques utilisant des immunogènes
23.
Display screen or portion thereof with transitional graphical user interface
In embodiments, a method includes receiving a first image comprising a cell boundary stain and determining a cellular boundary mask from the first image. The cellular boundary mask represents a first plurality of cellular boundaries. The method includes receiving a second image comprising a nuclear stain and determining a nuclear mask based on the second image. The nuclear mask representing a plurality of nuclei. The method further includes, after determining the cellular boundary mask, expanding at least some nuclei of the plurality of nuclei using one or more nuclear expansion models to obtain a second plurality of cellular boundaries. In the method, the second plurality of cellular boundaries is distinct from the first plurality of cellular boundaries.
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.
Methods for the design of a codebook comprising a set of codewords that are assigned to barcoded target analytes in a multiplexed in situ assay are described, where the codebook is designed to minimize the impact of target analyte spatial crowding on accurate decoding and detection of target analytes. Methods for performing in situ decoding using the disclosed codebooks, where, for all valid code words in the codebook, a first Hamming distance between a first logical bitwise OR combination of any pair of valid code words and a second logical bitwise OR combination of any other pair of valid code words is greater than or equal to 1, are also described.
Systems and methods are provided for targeting one or more features in a region of interest, and efficiently removing the features from an array of features. The systems and methods can remove one or more beads that contain analytes, intermediate agents, or a combination thereof, from the region of interest of a substrate by emitting light toward the beads on the substrate.
G01N 1/02 - Dispositifs pour prélever des échantillons
G01N 1/06 - Dispositifs pour prélever des échantillons à l'état solide, p. ex. par coupe à l'outil procurant une tranche mince, p. ex. "microtome"
G01N 1/28 - Préparation d'échantillons pour l'analyse
G01N 33/543 - Tests immunologiquesTests faisant intervenir la formation de liaisons biospécifiquesMatériaux à cet effet avec un support insoluble pour l'immobilisation de composés immunochimiques
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.
G16B 15/00 - TIC spécialement adaptées à l’analyse de structures moléculaires bidimensionnelles ou tridimensionnelles, p. ex. relations structurelles ou fonctionnelles ou alignement de structures
G16B 25/20 - Réaction en chaîne par polyméraseConception d’amorces ou de sondesOptimisation de la sonde
Provided herein are methods, compositions, and kits for determining the spatial location of target nucleic acids, including endogenous and exogenous target nucleic acids, in a biological sample using padlock probes and substrates with spatially barcoded capture probes. Also disclosed herein are methods for determining a presence and/or location of a microbe (e.g., archaea, fungi, bacteria) in a biological sample, e.g., by determining the presence and/or location of a microbial target nucleic acid in the biological sample.
Various embodiments of the present disclosure disclose methods and systems for actively monitoring an opto-fluidic instrument for vibrational disturbances and correcting images obtained during the detected disturbances. In various embodiments, an optical imaging system may acquire Z-stack images of samples supported by an XY-stage. Vibrations can cause individual stacks to be sheared, i.e., to not be co-located with respect to neighboring Z-stacks. In various embodiments, an offset between the measured positions and the expected positions of the sheared stacks may be computed, and a determination as to whether to correct or reacquire the Z-stack images may be made based at least in part on the computed offset.
H04N 23/68 - Commande des caméras ou des modules de caméras pour une prise de vue stable de la scène, p. ex. en compensant les vibrations du boîtier de l'appareil photo
32.
Display screen or portion thereof with graphical user interface
The present disclosure relates in some aspects to methods for analyzing target nucleic acids and their spatial locations in a biological sample using Argonaute proteins. In some aspects, a barcode probe library comprising a plurality of probes each comprising a plurality of barcode subunits that identifies a target analyte is detected in situ in the sample. Also provided are compositions and kits for use in accordance with the methods.
Featured are devices, systems, and methods of use for detecting binding of polynucleotide-peptide conjugate-major histocompatibility complex (pMHC) monomers to a T cell receptors (TCR) on a T cell, and the use of a peptide library to detect binding of antigenic peptides in a pMHC monomer to a T cell receptor (TCR) on a T cell.
G01N 33/569 - Tests immunologiquesTests faisant intervenir la formation de liaisons biospécifiquesMatériaux à cet effet pour micro-organismes, p. ex. protozoaires, bactéries, virus
In some embodiments described herein are methods performed in situ for analyzing chromatin interaction events in a cell or in cells of a sample such as a non-homogenized tissue sample. The methods can comprise the spatial analysis of chromatin interaction events across cell populations in a biological sample. The methods can further comprise obtaining a biological sample, hybridizing probes to target nucleic acid sequences involved in chromatin interaction events and producing a nucleic acid sequence comprising all or part of the target nucleic acid sequences, amplifying the nucleic acid sequence so produced and detecting the amplified nucleic acid sequence in situ.
C12Q 1/6818 - Tests d’hybridation caractérisés par les moyens de détection impliquant l’interaction de plusieurs marqueurs, p. ex. transfert d’énergie de résonance
C12Q 1/6837 - Couplage enzymatique ou biochimique d’acides nucléiques à une phase solide utilisant des réseaux de sondes ou des puces à sondes
36.
Display screen or portion thereof with transitional graphical user interface
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 or the biological sample, wherein the porous insert limits free flow space in the chamber, thereby reducing fluid convection adjacent to the biological sample.
Provided herein are methods for analyzing a biological sample that include contacting the biological sample with a non-conductive substrate; and contacting a matrix to a surface of the biological sample. Further methods optionally include performing mass spectrometry analysis; determining presence of a first analyte in the biological sample; and/or analyzing a second analyte on the surface of the biological sample to determine presence of the first and the second analyte in the biological sample.
Provided herein are methods of determining a location of a target analyte in a non-permeabilized biological sample and methods of reducing background binding of an analyte on an 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 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.
Compositions, kits, and methods for reducing mislocalization of analytes from a biological sample in the context of an array-based spatial analysis platform are disclosed herein. Also, disclosed herein are a first region of capture probes, where the capture probes include: (i) a spatial barcode, (ii) a first capture domain, and (iii) one or more functional domains, and a second region of capture probes, where the capture probes include a second capture domain. The second region of capture probes can capture analytes from portions of the biological sample that exceed the boundaries of the first region of capture probes, thereby reducing analyte mislocalization and improving the accuracy of the array-based spatial analysis platform.
The present disclosure provides chimeric ligase polypeptides comprising a ligase polypeptide domain operably linked to a catalytically inactive ribonuclease H (RNAseH) polypeptide domain; compositions, isolated nucleic acid, and vectors comprising the chimeric ligase polypeptides; and multiplexed methods for detecting nuclei acids in a biological sample using hybridization-based in situ sequencing assays.
A lid for forming a sealed fluidic chamber includes a cover having a planar outer surface and an inner surface separated from the planar outer surface by a thickness, and a skirt extending about a perimeter of the inner surface, where the skirt and the inner surface define a recess. The lid further includes a flange extending about a perimeter of the skirt, where the flange is parallel to the planar outer surface. The lid further includes a first crossbar and second crossbar extending parallel to the first and second side, respectively. The first crossbar and second crossbars have a first and second pair of snap joint elements, respectively.
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.
A lid for forming a sealed fluidic chamber includes a cover having a planar outer surface and an inner surface separated from the planar outer surface by a thickness, and a skirt extending about a perimeter of the inner surface, where the skirt and the inner surface define a recess. The lid further includes a flange extending about a perimeter of the skirt, where the flange is parallel to the planar outer surface. The lid further includes a first crossbar and second crossbar extending parallel to the first and second side, respectively. The first crossbar and second crossbars have a first and second pair of snap joint elements, respectively.
Provided herein are methods, systems, and computer program products for image fusion. A first z-stack of images of a biological sample may be received. The first z-stack of images may correspond to a first field of view, which comprises a plurality of patches. A second z- stack of images of the biological sample may be received. The second z-stack may correspond to the first field of view. A focus map may be determined based on the second z- stack, the focus map indicating, for each of a plurality of patches of the first field of view, one of the images of the second z-stack bringing into focus that patch of the first field of view. The focus map may be applied to the first and second z- stacks to generate respective first and second fused images. The first fused image may be subtracted from the second fused image to produce a subtracted image.
G06T 5/50 - Amélioration ou restauration d'image utilisant plusieurs images, p. ex. moyenne ou soustraction
G06T 7/33 - Détermination des paramètres de transformation pour l'alignement des images, c.-à-d. recalage des images utilisant des procédés basés sur les caractéristiques
G06T 7/35 - Détermination des paramètres de transformation pour l'alignement des images, c.-à-d. recalage des images utilisant des procédés statistiques
50.
PROFILING OF BIOLOGICAL ANALYTES WITH SPATIALLY BARCODED OLIGONUCLEOTIDE ARRAYS
This disclosure relates to methods for spatial profiling of analytes present in a biological sample. Also provided are methods for using spatially barcoded substrates to detect a biological analyte in a cell culture, an organism, and organoid. Also provided are methods for using spatially barcoded substrates to detect the temporal profile of a biological analyte.
C12Q 1/6874 - Méthodes de séquençage faisant intervenir des réseaux d’acides nucléiques, p. ex. séquençage par hybridation [SBH]
C12Q 1/6886 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour les maladies provoquées par des altérations du matériel génétique pour le cancer
G16B 30/10 - Alignement de séquenceRecherche d’homologie
Provided herein are methods, systems, and computer program products for image fusion. A first z-stack of images of a biological sample may be received. The first z-stack of images may correspond to a first field of view, which comprises a plurality of patches. A second z-stack of images of the biological sample may be received. The second z-stack may correspond to the first field of view. A focus map may be determined based on the second z-stack, the focus map indicating, for each of a plurality of patches of the first field of view, one of the images of the second z-stack bringing into focus that patch of the first field of view. The focus map may be applied to the first and second z-stacks to generate respective first and second fused images. The first fused image may be subtracted from the second fused image to produce a subtracted image.
G06T 5/50 - Amélioration ou restauration d'image utilisant plusieurs images, p. ex. moyenne ou soustraction
G06T 3/4007 - Changement d'échelle d’images complètes ou de parties d’image, p. ex. agrandissement ou rétrécissement basé sur l’interpolation, p. ex. interpolation bilinéaire
G06T 5/20 - Amélioration ou restauration d'image utilisant des opérateurs locaux
G06T 7/194 - DécoupageDétection de bords impliquant une segmentation premier plan-arrière-plan
G06T 7/33 - Détermination des paramètres de transformation pour l'alignement des images, c.-à-d. recalage des images utilisant des procédés basés sur les caractéristiques
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.
C12Q 1/6874 - Méthodes de séquençage faisant intervenir des réseaux d’acides nucléiques, p. ex. séquençage par hybridation [SBH]
C12Q 1/6876 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes
C12Q 1/6881 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour le typage de tissu ou de cellule, p. ex. sondes d’antigène leucocytaire humain [HLA]
G01N 33/483 - Analyse physique de matériau biologique
G01N 35/00 - Analyse automatique non limitée à des procédés ou à des matériaux spécifiés dans un seul des groupes Manipulation de matériaux à cet effet
G02B 21/16 - Microscopes adaptés pour éclairage ultraviolet
G02B 21/26 - PlatinesMoyens de réglage pour celles-ci
G02B 21/34 - Lames de microscope, p. ex. montage d'échantillons sur des lames de microscope
G02B 21/36 - Microscopes aménagés pour la photographie ou la projection
The present disclosure provides methods, systems, and compositions for parallel processing of nucleic acid samples. Methods and systems of the present disclosure comprise the use of sample-specific barcode sequences, which facilitate the multiplexing of samples, detection of discrete cell populations within a pooled population, and detection of partitions comprising more than one cell.
C12Q 1/6886 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour les maladies provoquées par des altérations du matériel génétique pour le cancer
B01L 3/00 - Récipients ou ustensiles pour laboratoires, p. ex. verrerie de laboratoireCompte-gouttes
C12M 3/08 - Appareils pour la désagrégation des tissus
C12N 5/10 - Cellules modifiées par l'introduction de matériel génétique étranger, p. ex. cellules transformées par des virus
C12N 15/10 - Procédés pour l'isolement, la préparation ou la purification d'ADN ou d'ARN
C12P 19/34 - Polynucléotides, p. ex. acides nucléiques, oligoribonucléotides
C12Q 1/6804 - Analyse d’acides nucléiques utilisant des immunogènes
C12Q 1/6806 - Préparation d’acides nucléiques pour analyse, p. ex. pour test de réaction en chaîne par polymérase [PCR]
C12Q 1/6881 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour le typage de tissu ou de cellule, p. ex. sondes d’antigène leucocytaire humain [HLA]
G16B 30/00 - TIC spécialement adaptées à l’analyse de séquences impliquant des nucléotides ou des aminoacides
G16B 50/00 - TIC pour la programmation d’outils ou de systèmes de bases de données spécialement adaptées à la bio-informatique
54.
Display screen or portion thereof with transitional graphical user interface
The present disclosure relates in some aspects to methods, systems, and kits for sequencing a template nucleic acid molecule using click chemistry bioconjugation. In some aspects, a coupling reaction is performed between a first functional group of a modified nucleotide molecule and a second functional group of a modified 3′ reversibly terminated nucleotide on a priming strand. In some aspects, this coupling reaction creates stable retention of the fluorescent read-out.
An adapter for incorporation into an open well flow cell, the adapter including a body having an upper surface, a lower surface spaced from the upper surface defining a thickness profile therebetween, at least one side extending about a perimeter of the body, an inlet formed in the body extending through the thickness of the upper surface and the lower surface, at least one foot extending from the lower surface, each foot of the at least one foot extending a vertical distance away from the lower surface and a protrusion extending from the upper surface. The adaptor configured to be placed over, or onto, an open well flow cell to form an effectively closed, and reversible, flow cell. A variety of structural features are included to inhibit/prohibit gas bubbles from evaporating from the fluid reagent within the cell, and/or providing egress or venting of such bubbles.
The present disclosure relates in some aspects to methods, systems, and kits for sequencing a template nucleic acid molecule using nucleotide-fluorophore complexes. In some embodiments, each nucleotide-fluorophore complex comprises: (i) a single nucleotide attached to a core; and (ii) a plurality of fluorophores attached to the core; wherein each nucleotide-fluorophore complex comprises no more than one nucleotide. In some embodiments, the methods provided herein achieve improved signal intensity and sensitivity.
Provided herein are methods of sequencing comprising click chemistry bioconjugation. In some embodiments, target polynucleotide sequences on the same or different molecules are contacted with and hybridize to probes comprising click functional groups. Probes (e.g., reading probes) hybridizing to adaptor sequences adjacent to different sequences of interest (e.g., barcodes to be sequenced) can be hybridized simultaneously in large pools. In some embodiments, the provided methods achieve multiplexing without requiring separate hybridization of probes (e.g., reading probes) for each sequencing-by-ligation cycle, thereby reducing total hybridization time which is typically a most time-consuming step in in situ technologies. In some aspects, the hybridized probes (e.g., reading probes) are clicked onto detectable probes to analyze a sequence of a target polynucleotide in a sequencing-by-ligation fashion.
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.
An adapter for incorporation into an open well flow cell, the adapter including a body having an upper surface, a lower surface spaced from the upper surface defining a thickness profile therebetween, at least one side extending about a perimeter of the body, an inlet formed in the body extending through the thickness of the upper surface and the lower surface, at least one foot extending from the lower surface, each foot of the at least one foot extending a vertical distance away from the lower surface and a protrusion extending from the upper surface. The adaptor configured to be placed over, or onto, an open well flow cell to form an effectively closed, and reversible, flow cell. A variety of structural features are included to inhibit/prohibit gas bubbles from evaporating from the fluid reagent within the cell, and/or providing egress or venting of such bubbles.
Systems and methods are provided for stabilization of cells and retention of biomolecules therein for further analysis. Block copolymers are inserted into membranes of biological particles, cell membranes or organelles (e.g., organelle membranes) and polymerized to form an intra-biological particle (e.g., intracellular) polymer network.
C08L 53/00 - Compositions contenant des copolymères séquencés possédant au moins une séquence d'un polymère obtenu par des réactions ne faisant intervenir que des liaisons non saturées carbone-carboneCompositions contenant des dérivés de tels polymères
A61K 47/60 - Préparations médicinales caractérisées par les ingrédients non actifs utilisés, p. ex. les supports ou les additifs inertesAgents de ciblage ou de modification chimiquement liés à l’ingrédient actif l’ingrédient non actif étant chimiquement lié à l’ingrédient actif, p. ex. conjugués polymère-médicament l’ingrédient non actif étant un agent de modification l’agent de modification étant un composé organique macromoléculaire, p. ex. une molécule oligomérique, polymérique ou dendrimérique obtenu par des réactions autres que celles faisant intervenir uniquement des liaisons non saturées carbone-carbone, p. ex. polyurées ou polyuréthanes le composé organique macromoléculaire étant un oligomère, un polymère ou un dendrimère de polyoxyalkylène, p. ex. PEG, PPG, PEO ou polyglycérol
A61K 47/69 - Préparations médicinales caractérisées par les ingrédients non actifs utilisés, p. ex. les supports ou les additifs inertesAgents de ciblage ou de modification chimiquement liés à l’ingrédient actif l’ingrédient non actif étant chimiquement lié à l’ingrédient actif, p. ex. conjugués polymère-médicament le conjugué étant caractérisé par sa forme physique ou sa forme galénique, p. ex. émulsion, particule, complexe d’inclusion, stent ou kit
C08G 65/329 - Polymères modifiés par post-traitement chimique avec des composés organiques
Systems and methods are provided for targeting one or more features in a region of interest, and efficiently removing the features from an array of features. The systems and methods can remove one or more beads that contain analytes, intermediate agents, or a combination thereof, from the region of interest of a substrate by emitting light toward the beads on the substrate.
G01N 1/02 - Dispositifs pour prélever des échantillons
G01N 1/06 - Dispositifs pour prélever des échantillons à l'état solide, p. ex. par coupe à l'outil procurant une tranche mince, p. ex. "microtome"
G01N 1/28 - Préparation d'échantillons pour l'analyse
G01N 33/543 - Tests immunologiquesTests faisant intervenir la formation de liaisons biospécifiquesMatériaux à cet effet avec un support insoluble pour l'immobilisation de composés immunochimiques
In some aspects disclosed herein are methods and compositions for detecting a target nucleic acid molecule, said method comprising performing a linear oligo hybridization chain reaction (LO-HCR) to generate a polymeric product, and detecting the polymeric product, thereby detecting the target nucleic acid molecule.
Devices, systems, and their methods of use, for generating and collecting droplets are provided. The device includes a collection region comprising a side wall canted at an angle. The invention further provides multiplex devices that increase droplet formation.
B01F 25/314 - Mélangeurs à injecteurs dans des conduits ou des tubes dans lesquels circule le composant principal dans lesquels des composants supplémentaires sont introduits à la circonférence du conduit
B01F 33/3011 - Micromixeurs utilisant des moyens spécifiques pour disposer les écoulements à mélanger, p. ex. des géométries ou des dispositions de canaux utilisant un courant de gainage d'un fluide entourant un courant central d'un autre fluide, p. ex. pour réduire la section transversale du courant central ou pour produire des gouttelettes à partir du courant central
G01N 15/10 - Recherche de particules individuelles
G01N 15/14 - Techniques de recherche optique, p. ex. cytométrie en flux
G01N 15/149 - Techniques de recherche optique, p. ex. cytométrie en flux spécialement adaptées au tri des particules, p. ex. selon leur taille ou leurs propriétés
G01N 15/1492 - Techniques de recherche optique, p. ex. cytométrie en flux spécialement adaptées au tri des particules, p. ex. selon leur taille ou leurs propriétés dans des gouttelettes
65.
INCREASING EFFICIENCY OF SPATIAL ANALYSIS IN A BIOLOGICAL SAMPLE
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 for analyzing target nucleic acids and their spatial locations in a biological sample. In some aspects, the presence/absence, amount, and/or identity of variant sequences (e.g., single nucleotide variations such as SNPs or point mutations) in a plurality of target nucleic acids in a cell or tissue sample are analyzed in situ in the sample. Also provided are compositions and kits for use in accordance with the methods.
Provided herein are computer-implemented methods including receiving, by a processor, a data set comprising cellular data, and presenting an end user with a visualization tool, wherein the cellular data may include at least one of an antigen binding specificity value associated with a cell receptor and a unique molecular identifier (UMI) count associated with the cell receptor. The visualization tool can provide a dynamic display of the data set by generating a visual sequence from the data set, displaying at least a portion of the generated visual sequence, and displaying in response to a user interaction with the visualization tool, the antigen binding specificity value or the UMI associated with the cell receptor in conjunction with the at least a portion of the visual sequence. The visual sequences include sets of indicia corresponding paired chains of the cell receptor.
Devices, methods, and kits for generating droplets are provided. The devices, methods, and kits are designed to reduce the effects of settling of particles during droplet production.
Provided herein are method for determining a location of nucleic acids in fixed biological samples, in which the method includes use of a template switching oligonucleotide (TSO) and a randomer during second strand synthesis.
The present disclosure in some aspects relates to methods, compositions, and kits for rolling circle amplification (RCA) comprising extending a nucleic acid priming sequence hybridized to a circular nucleic acid template using a polymerase to generate an extended priming sequence, wherein the circular nucleic acid template is crosslinked to a second nucleic acid strand; de-crosslinking the circular nucleic acid template from the second nucleic acid strand; and extending the extended priming sequence to generate a rolling circle amplification product (RCP).
The present disclosure relates in some aspects to methods, systems, and kits for analyzing a biological sample comprising generating a rolling circle amplification product (RCP) using a target ribonucleic acid (RNA) as a primer. In some aspects, RNase H and a nucleic acid oligonucleotide are used to generate a free 3′ end of the target RNA to prime RCA.
The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing and analyte characterization. Such polynucleotide processing may be useful for a variety of applications, including analyte characterization by polynucleotide sequencing. 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 including, for example, protein (e.g., cell surface or intracellular proteins), genomic DNA, and RNA (e.g., mRNA or CRISPR guide RNAs). Also described herein, are barcoded labelling agents and oligonucleotide molecules useful for “tagging” analytes for characterization.
C12Q 1/6874 - Méthodes de séquençage faisant intervenir des réseaux d’acides nucléiques, p. ex. séquençage par hybridation [SBH]
C12Q 1/6806 - Préparation d’acides nucléiques pour analyse, p. ex. pour test de réaction en chaîne par polymérase [PCR]
C12Q 1/683 - Tests d’hybridation pour la détection de mutation ou de polymorphisme faisant intervenir des enzymes de restriction, p. ex. polymorphisme de longueur de fragment de resctriction
74.
COMPOSITIONS AND METHODS FOR ANALYZING GENOMIC INSERTION SITES OF EXOGENOUS NUCLEIC ACIDS
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.
Methods and compositions for analyzing a library comprising a plurality of amplicons comprising identifier sequences are provided, for example, a library of amplicons in a cell or tissue sample attached to a solid support. For example, amplicons are sequenced using a polymerase to incorporate a plurality of cognate nucleotides into the sequencing primer or an extension product thereof to generate a plurality of extension products.
C12Q 1/6874 - Méthodes de séquençage faisant intervenir des réseaux d’acides nucléiques, p. ex. séquençage par hybridation [SBH]
C12N 15/10 - Procédés pour l'isolement, la préparation ou la purification d'ADN ou d'ARN
C12Q 1/48 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir une transférase
The present disclosure relates in some aspects to methods, systems, and kits for sequencing a template nucleic acid molecule, where the methods comprise: (i) contacting a priming strand bound to the template nucleic acid molecule with a first plurality of nucleotide molecules and a polymerase coupled to a heterologous polynucleotide-binding moiety to form a complex comprising a 3′ terminus of the priming strand, the template nucleic acid molecule, the polymerase, and a nucleotide molecule of the first plurality of nucleotide molecules, wherein the polynucleotide-binding moiety enhances stability of the complex, and wherein the priming strand comprises a reversibly-terminated nucleotide at its 3′ end such that the nucleotide molecule of the transient complex is not incorporated; and (ii) detecting a presence of the nucleotide molecule in the complex to identify a complementary nucleotide in the template nucleic acid molecule.
C12Q 1/6874 - Méthodes de séquençage faisant intervenir des réseaux d’acides nucléiques, p. ex. séquençage par hybridation [SBH]
C12Q 1/48 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir une transférase
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.
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 tissue samples (e.g., antibodies produced by B cells in tumor tissue samples or TCRs produced by T cells in tumor tissue samples) using spatial methodologies, and for the production and characterization of recombinant antigen-binding molecules (e.g., antibodies, TCRs) with desired properties.
G01N 33/68 - Analyse chimique de matériau biologique, p. ex. de sang ou d'urineTest par des méthodes faisant intervenir la formation de liaisons biospécifiques par ligandsTest immunologique faisant intervenir des protéines, peptides ou amino-acides
C12N 15/10 - Procédés pour l'isolement, la préparation ou la purification d'ADN ou d'ARN
80.
METHODS, COMPOSITIONS, AND KITS FOR DETERMINING THE LOCATION OF AN ANALYTE IN A BIOLOGICAL SAMPLE
Provided herein are assemblies, systems, and methods for increasing the effective stiffness and/or damping of a pneumatic vibration isolation system. In various embodiments, a system includes a payload to be isolated from vibrations, a source of pressurized gas, a plurality of pneumatic vibration damping modules coupled to the payload, and a one-way restrictor valve fluidically coupled between the source of pressurized gas and the plurality of vibration damping modules. Each pneumatic vibration damping module of the plurality of pneumatic vibration damping modules is fluidically coupled to the source of pressurized gas.
F16F 15/023 - Suppression des vibrations dans les systèmes non rotatifs, p. ex. dans des systèmes alternatifsSuppression des vibrations dans les systèmes rotatifs par l'utilisation d'organes ne se déplaçant pas avec le système rotatif utilisant des moyens fluides
F16F 15/027 - Suppression des vibrations dans les systèmes non rotatifs, p. ex. dans des systèmes alternatifsSuppression des vibrations dans les systèmes rotatifs par l'utilisation d'organes ne se déplaçant pas avec le système rotatif utilisant des moyens fluides comprenant des dispositifs de commande
F16F 9/04 - Ressorts, amortisseurs de vibrations, amortisseurs de chocs ou amortisseurs de mouvement de structure similaire, utilisant un fluide ou moyen équivalent comme agent d'amortissement utilisant un gaz uniquement dans une chambre à paroi flexible
82.
METHODS OF CRISPR GUIDE DETECTION IN SINGLE CELL WORKFLOWS VIA RNA-TEMPLATED 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.
Provided herein are systems and methods for analyzing biomolecules (e.g., nucleic acid molecules, proteins). A method of nucleic acid analysis can comprise: (a) providing a sample comprising a cell comprising a target polynucleotide comprising a first exon segment and a second exon segment, wherein the first exon segment and the second exon segment flank opposite ends of a splice junction site of the target polynucleotide. The method can further comprise (b) contacting the cell with: (i) a first probe, wherein the first probe hybridizes to a first target sequence of the first exon segment, and (ii) a second probe, wherein the second probe hybridizes to a second target sequence of the second exon segment. The method can further comprise (c) linking the first probe and the second probe together, thereby generating a probe-linked nucleic acid molecule comprising the first probe and the second probe. The method can further comprise (d) identifying a sequence of the probe-linked nucleic acid molecule or derivative thereof, thereby locating the splice junction site of the target polynucleotide.
Provided herein are systems and methods for analyzing biomolecules (e.g., nucleic acid molecules, proteins). A method of nucleic acid analysis can comprise: (a) providing a sample comprising a cell comprising a target polynucleotide comprising a first exon segment and a second exon segment, wherein the first exon segment and the second exon segment flank opposite ends of a splice junction site of the target polynucleotide. The method can further comprise (b) contacting the cell with: (i) a first probe, wherein the first probe hybridizes to a first target sequence of the first exon segment, and (ii) a second probe, wherein the second probe hybridizes to a second target sequence of the second exon segment. The method can further comprise (c) linking the first probe and the second probe together, thereby generating a probe-linked nucleic acid molecule comprising the first probe and the second probe. The method can further comprise (d) identifying a sequence of the probe-linked nucleic acid molecule or derivative thereof, thereby locating the splice junction site of the target polynucleotide.
Provided herein are methods and compositions for sequencing gRNAs. The methods and compositions are compatible with single-cell sequencing workflows, including in combination with analysis of additional non-gRNA analytes such as cellular transcripts. The methods provided herein can facilitate high-resolution phenotypic analysis in large-scale CRISPR/Cas-based screens.
Provided herein are assemblies, systems, and methods for increasing the effective stiffness and/or damping of a pneumatic vibration isolation system. In various embodiments, a system includes a payload to be isolated from vibrations, a source of pressurized gas, a plurality of pneumatic vibration damping modules coupled to the payload, and a one-way restrictor valve fluidically coupled between the source of pressurized gas and the plurality of vibration damping modules. Each pneumatic vibration damping module of the plurality of pneumatic vibration damping modules is fluidically coupled to the source of pressurized gas.
F16F 15/023 - Suppression des vibrations dans les systèmes non rotatifs, p. ex. dans des systèmes alternatifsSuppression des vibrations dans les systèmes rotatifs par l'utilisation d'organes ne se déplaçant pas avec le système rotatif utilisant des moyens fluides
F16F 15/00 - Suppression des vibrations dans les systèmesMoyens ou dispositions pour éviter ou réduire les forces de déséquilibre, p. ex. dues au mouvement
F16F 15/027 - Suppression des vibrations dans les systèmes non rotatifs, p. ex. dans des systèmes alternatifsSuppression des vibrations dans les systèmes rotatifs par l'utilisation d'organes ne se déplaçant pas avec le système rotatif utilisant des moyens fluides comprenant des dispositifs de commande
87.
METHODS AND SYSTEMS FOR PROCESSING POLYNUCLEOTIDES
The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing and analyte characterization. Such polynucleotide processing may be useful for a variety of applications, including analyte characterization by polynucleotide sequencing. 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 including, for example, protein (e.g., cell surface or intracellular proteins), genomic DNA, and RNA (e.g., mRNA or CRISPR guide RNAs). Also described herein, are barcoded labelling agents and oligonucleotide molecules useful for “tagging” analytes for characterization.
C12Q 1/6874 - Méthodes de séquençage faisant intervenir des réseaux d’acides nucléiques, p. ex. séquençage par hybridation [SBH]
C12Q 1/6806 - Préparation d’acides nucléiques pour analyse, p. ex. pour test de réaction en chaîne par polymérase [PCR]
C12Q 1/683 - Tests d’hybridation pour la détection de mutation ou de polymorphisme faisant intervenir des enzymes de restriction, p. ex. polymorphisme de longueur de fragment de resctriction
88.
Systems and methods for image segmentation using multiple stain indicators
In embodiments, a method includes reading a nuclear segmentation mask of an image including pixels arranged in two dimensions. The nuclear segmentation mask identifies cellular nuclei stained with a cellular nucleus stain. The method includes determining amplitudes for each pixel. Each amplitude corresponds to exactly one dimension of the two dimensions. The method includes constructing a graph having nodes and edges. Each node corresponds to a pixel. Nodes corresponding to neighboring pixels are connected by an edge. The method includes assigning a weight to each edge. Each edge's weight is based on amplitudes of pixels corresponding to nodes connected thereby. The method further includes, based on the graph, determining, for each cellular nuclei, a heat map corresponding to a predicted cell region associated with that cellular nucleus. The method further includes based on the heat maps of the cellular nuclei, determining a cellular segmentation mask comprising predicted cell regions.
The disclosure provides systems and methods for droplet processing. For example, a method can include providing a first droplet and a second droplet. In some cases, the first droplet may have a first concentration of a reagent and the second droplet may have a second concentration of the reagent. The second droplet may comprise a bead or a biological particle. The method can also include subjecting the first droplet and the second droplet to conditions sufficient to transfer the reagent from the first droplet to the second droplet, thereby decreasing the first concentration in the first droplet and increasing the second concentration in the second droplet.
C12Q 1/6883 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour les maladies provoquées par des altérations du matériel génétique
C12Q 1/6827 - Tests d’hybridation pour la détection de mutation ou de polymorphisme
G16B 30/00 - TIC spécialement adaptées à l’analyse de séquences impliquant des nucléotides ou des aminoacides
G16B 30/10 - Alignement de séquenceRecherche d’homologie
In embodiments, a method includes reading a nuclear segmentation mask of an image including pixels arranged in two dimensions. The nuclear segmentation mask identifies cellular nuclei stained with a cellular nucleus stain. The method includes determining amplitudes for each pixel. Each amplitude corresponds to exactly one dimension of the two dimensions. The method includes constructing a graph having nodes and edges. Each node corresponds to a pixel. Nodes corresponding to neighboring pixels are connected by an edge. The method includes assigning a weight to each edge. Each edge's weight is based on amplitudes of pixels corresponding to nodes connected thereby. The method further includes, based on the graph, determining, for each cellular nuclei, a heat map corresponding to a predicted cell region associated with that cellular nucleus. The method further includes based on the heat maps of the cellular nuclei, determining a cellular segmentation mask comprising predicted cell regions.
G06T 7/162 - DécoupageDétection de bords impliquant des procédés basés sur des graphes
G06T 7/187 - DécoupageDétection de bords impliquant des croissances de zonesDécoupageDétection de bords impliquant des fusions de zonesDécoupageDétection de bords impliquant un étiquetage de composantes connexes
91.
NUCLEIC ACID PROBE SETS COMPRISING STEM REGION FOR SAMPLE ANALYSIS
The present disclosure relates, in some aspects, to methods and compositions for analyzing a biological sample. In some aspects, the methods comprise use of a probe set comprising a first and second polynucleotide which hybridize to a target nucleic acid in the biological sample and form a circularized probe. In some aspects, the methods and compositions provided herein improve the detection of nucleic acids in a biological sample.
Methods and compositions for performing a rolling circle amplification (RCA) reaction using circularized template comprising identifier sequences are provided. Sequencing is performed on the RCA product using a polymerase to incorporate a plurality of cognate nucleotides into the sequencing primer or an extension product thereof to generate an extension product.
Provided herein are methods, compositions, and kits to spatially detect a polypeptide-nucleic acid complex or a protein-nucleic acid complex in a biological sample. For example, such methods can include contacting binding agents with a biological sample, wherein a binding agent specifically binds a polypeptide of a polypeptide-nucleic acid complex; aligning the biological sample with a substrate comprising a plurality of capture moieties, wherein the binding agent interacts with a capture moiety; releasing the nucleic acid of the binding agent-polypeptide-nucleic acid complex; hybridizing the released nucleic acid to a capture domain of a capture probe on an array; and using determined sequences of a spatial barcode in the capture probe and the released nucleic acid to determine the location of the released nucleic acid in the biological sample, thereby determining the location of the polypeptide-nucleic acid complex in the biological sample.
C12Q 1/6874 - Méthodes de séquençage faisant intervenir des réseaux d’acides nucléiques, p. ex. séquençage par hybridation [SBH]
C12Q 1/37 - Procédés de mesure ou de test faisant intervenir des enzymes, des acides nucléiques ou des micro-organismesCompositions à cet effetProcédés pour préparer ces compositions faisant intervenir une hydrolase faisant intervenir une peptidase ou une protéinase
C12Q 1/6804 - Analyse d’acides nucléiques utilisant des immunogènes
The application provides compositions including engineered reverse transcriptases with at least one altered reverse-transcriptase related activity. The engineered 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.
The present disclosure relates to methods, compositions and systems for haplotype phasing and copy number variation assays. Included within this disclosure are methods and systems for combining the barcode comprising beads with samples in multiple separate partitions, as well as methods of processing, sequencing and analyzing barcoded samples.
C12Q 1/6883 - Produits d’acides nucléiques utilisés dans l’analyse d’acides nucléiques, p. ex. amorces ou sondes pour les maladies provoquées par des altérations du matériel génétique
C12Q 1/6827 - Tests d’hybridation pour la détection de mutation ou de polymorphisme
G16B 30/00 - TIC spécialement adaptées à l’analyse de séquences impliquant des nucléotides ou des aminoacides
G16B 30/10 - Alignement de séquenceRecherche d’homologie
The present disclosure relates to compositions and methods for reversible fixation of biological samples using fixation reagents that form bis-carbamate crosslinks between amine-bearing moieties in biomolecules.
C07D 403/12 - Composés hétérocycliques contenant plusieurs hétérocycles, comportant des atomes d'azote comme uniques hétéro-atomes du cycle, non prévus par le groupe contenant deux hétérocycles liés par une chaîne contenant des hétéro-atomes comme chaînons
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.
Provided herein are methods, compositions, and kits to spatially detect genetic variants in a target nucleic acid via templated-ligation and reversible terminator nucleotides.
