Abstract: Compositions, methods and kits are disclosed for high-sensitivity single molecule digital counting by the stochastic labeling of a collection of identical molecules by attachment of a diverse set of labels. Each copy of a molecule randomly chooses from a non-depleting reservoir of diverse labels. Detection may be by a variety of methods including hybridization based or sequencing. Molecules that would otherwise be identical in information content can be labeled to create a separately detectable product that is unique or approximately unique in a collection. This stochastic transformation relaxes the problem of counting molecules from one of locating and identifying identical molecules to a series of binary digital questions detecting whether preprogrammed labels are present. The methods may be used, for example, to estimate the number of separate molecules of a given type or types within a sample.

Abstract: The disclosure provides compositions, methods, and systems for implementation of highly multiplexed molecular diagnostic assays involving color combinatorics, stimulus-responsive probes, tandem probes, conjugated polymer probes, and other mechanisms for increasing the number of targets that can be simultaneously detected in a digital assay. Multiplexed detection of targets is achieved in a rapid manner, with respect to sample partitioning and target detection using multiple color channels for detection. Implementation of methods described also achieve detection with significantly improved signal-to-noise ratio (SNR) values.

Abstract: Systems, methods, and compositions for generating a high-resolution spatial map of a distribution of targets of a sample are described. Processes for generating the spatial map can include: receiving the sample at a substrate having a distribution of functionalized particles, each having a stochastic barcode sequence paired with a position on the substrate; promoting interactions between the distribution of targets of the sample and the distribution of functionalized particles upon transmitting heat to a surface of the substrate opposite the distribution of functionalized particles; applying a set of reactions to the sample at the substrate, obtaining a set of sequences of a population of molecules generated from the set of reactions, the set of sequences associated with the distribution of targets labeled using the stochastic barcode sequences of the distribution of functionalized particles, and returning a set of positions of the distribution of targets upon processing the set of sequences.

Abstract: The disclosure provides compositions, methods, and systems for implementation of highly multiplexed molecular diagnostic assays involving color combinatorics, stimulus-responsive probes, tandem probes, conjugated polymer probes, and other mechanisms for increasing the number of targets that can be simultaneously detected in a digital assay. Multiplexed detection of targets is achieved in a rapid manner, with respect to sample partitioning and target detection using multiple color channels for detection. Implementation of methods described also achieve detection with significantly improved signal-to-noise ratio (SNR) values.

Abstract: Compositions, methods and kits are disclosed for high-sensitivity single molecule digital counting by the stochastic labeling of a collection of identical molecules by attachment of a diverse set of labels. Each copy of a molecule randomly chooses from a non-depleting reservoir of diverse labels. Detection may be by a variety of methods including hybridization based or sequencing. Molecules that would otherwise be identical in information content can be labeled to create a separately detectable product that is unique or approximately unique in a collection. This stochastic transformation relaxes the problem of counting molecules from one of locating and identifying identical molecules to a series of binary digital questions detecting whether preprogrammed labels are present. The methods may be used, for example, to estimate the number of separate molecules of a given type or types within a sample.

Abstract: This disclosure provides for devices, methods, and systems for generating a plurality of droplets within a collecting container at an extremely high rate (e.g., of at least 1 million droplets per minute, etc.), the plurality of droplets generated from an aqueous mixture comprising a set of single cells and a set of functionalized particles configured for a single cell assay. Upon generation, the plurality of droplets can be stabilized in position within a region of the collecting container, thereby providing a single-tube workflow for single cell analyses. Further, compositions implemented are structured to allow for overloading of partitions with functionalized particles, such that partitioned single-cells are co-localized with a subset of functionalized particles in a manner that allows for discernable tagging and downstream analyses.

Abstract: This disclosure provides for devices, methods, and systems for generating a plurality of droplets within a collecting container at an extremely high rate (e.g., of at least 1 million droplets per minute, etc.), the plurality of droplets generated from an aqueous mixture comprising a set of single cells and a set of functionalized particles configured for a single cell assay. Upon generation, the plurality of droplets can be stabilized in position within a region of the collecting container, thereby providing a single-tube workflow for single cell analyses. Further, compositions implemented are structured to allow for overloading of partitions with functionalized particles, such that partitioned single-cells are co-localized with a subset of functionalized particles in a manner that allows for discernable tagging and downstream analyses.

Abstract: The disclosure provides for methods, compositions, and kits for multiplex nucleic acid analysis of single cells. The methods, compositions and systems may be used for massively parallel single cell sequencing. The methods, compositions and systems may be used to analyze thousands of cells concurrently. The thousands of cells may comprise a mixed population of cells (e.g., cells of different types or subtypes, different sizes).

Abstract: The inventions cover systems and methods for generation of emulsions having suitable clarity without requiring refractive index matching between emulsion components. Systems can include: a substrate including a set of openings; a reservoir facing the substrate at a first side and containing a sample fluid configured for droplet formation upon interacting with the set of openings of the substrate; and a collecting container facing the substrate at a second side and containing a set of fluid layers configured with a density gradient and suitable immiscibility characteristics. One or more components of the system(s) can support methods for emulsion generation, in relation to enabling interactions between multiple continuous phases and a dispersed droplet phase to generate clear emulsions. Applications of the inventions(s) can include performance of droplet-based digital PCR in an improved manner (e.g., without requiring implementation of correction factors based upon Poisson statistics).

Abstract: The disclosure provides for methods, compositions, and kits for multiplex nucleic acid analysis of single cells. The methods, compositions and systems may be used for massively parallel single cell sequencing. The methods, compositions and systems may be used to analyze thousands of cells concurrently. The thousands of cells may comprise a mixed population of cells (e.g., cells of different types or subtypes, different sizes).

Abstract: The disclosure provides compositions, methods, and systems for extremely accurate generation of nucleic acid libraries, without use of bulk amplification methods. Accurate library preparation is achieved in a rapid manner, with respect to sample partitioning and amplification in a manner that achieves high performance in relation to low levels of amplification bias and low levels of artifact/chimeric sequence generation. Implementation of methods described also achieve library preparation with significantly reduced false positive rates, across a wide variety of applications.

Abstract: The invention(s) cover a composition, where units of the composition are configured to interact with each other (e.g., as neighbors) in order enable decoding of positions of captured target material relative to neighboring units of the composition. In embodiments, the composition includes: a body; and a set of molecules coupled to the body, the set of molecules comprising a first subset and a second subset, wherein the first subset is structured for target analyte capture, and wherein the second subset is structured for interactions with one or more neighboring objects. The invention(s) also cover systems incorporating one or more units of the composition and methods implementing units of the composition.

Abstract: The disclosure provides compositions, methods, and systems for implementation of highly multiplexed molecular diagnostic assays involving color combinatorics, stimulus-responsive probes, tandem probes, conjugated polymer probes, and other mechanisms for increasing the number of targets that can be simultaneously detected in a digital assay. Multiplexed detection of targets is achieved in a rapid manner, with respect to sample partitioning and target detection using multiple color channels for detection. Implementation of methods described also achieve detection with significantly improved signal-to-noise ratio (SNR) values.

Abstract: Methods, kits and systems are disclosed for analyzing one or more molecules in a sample. Analyzing the one or more molecules may comprise quantitation of the one or more molecules. Individual molecules may quantitated by PCR, arrays, beads, emulsions, droplets, or sequencing. Quantitation of individual molecules may further comprise stochastic labeling of the one or more molecules with a plurality of oligonucleotide tags to produce one or more stochastically labeled molecules. The methods may further comprise amplifying, sequencing, detecting, and/or quantifying the stochastically labeled molecules. The molecules may be DNA, RNA and/or proteins.

Abstract: The invention(s) cover a composition, where units of the composition are configured to interact with each other (e.g., as neighbors) in order enable decoding of positions of captured target material relative to neighboring units of the composition. In embodiments, the composition includes: a body; and a set of molecules coupled to the body, the set of molecules comprising a first subset and a second subset, wherein the first subset is structured for target analyte capture, and wherein the second subset is structured for interactions with one or more neighboring objects. The invention(s) also cover systems incorporating one or more units of the composition and methods implementing units of the composition.

Abstract: The disclosure provides for methods, compositions, and kits for multiplex nucleic acid analysis of single cells. The methods, compositions and systems may be used for massively parallel single cell sequencing. The methods, compositions and systems may be used to analyze thousands of cells concurrently. The thousands of cells may comprise a mixed population of cells (e.g., cells of different types or subtypes, different sizes).

Abstract: The present disclosure provide compositions, methods and kits for generating a set of combinatorial barcodes, and uses thereof for barcoding samples such as single cells or genomic DNA fragments. Some embodiments disclosed herein provide compositions comprising a set of component barcodes for producing a set of combinatorial barcodes. The set of component barcodes can comprise, for example, n×m unique component barcodes, wherein n and m are integers, each of the component barcodes comprises: one of n unique barcode subunit sequences; and one or two linker sequences or the complements thereof, wherein the component barcodes are configured to connect to each other through the one or two linker sequences or the complements thereof to produce a set of combinatorial barcodes.

Abstract: This disclosure provides for devices, methods, and systems for performing a non-invasive prenatal testing (NIPT) digital assay upon generating at least a large number of counts per chromosome for a set of chromosomes present in a sample, where performing the NIPT digital assay can include: distributing nucleic acids of the sample and materials for an amplification reaction across a plurality of partitions; amplifying the nucleic acids with the materials, within the plurality of partitions; and generating counts per chromosome upon detecting signals from the plurality of partitions. The inventions enable processing of samples for NIPT digital analyses and/or other digital analyses involving other loci of interest, with unprecedented partitioning, reaction, readout, and analytical performance.

Abstract: Systems, methods, and compositions for generating a high-resolution spatial map of a distribution of targets of a sample are described. Processes for generating the spatial map can include: receiving the sample at a substrate having a distribution of functionalized particles, each having a stochastic barcode sequence paired with a position on the substrate; promoting interactions between the distribution of targets of the sample and the distribution of functionalized particles upon transmitting heat to a surface of the substrate opposite the distribution of functionalized particles; applying a set of reactions to the sample at the substrate, obtaining a set of sequences of a population of molecules generated from the set of reactions, the set of sequences associated with the distribution of targets labeled using the stochastic barcode sequences of the distribution of functionalized particles, and returning a set of positions of the distribution of targets upon processing the set of sequences.

Abstract: The inventions cover systems and methods for generation of emulsions having suitable clarity without requiring refractive index matching between emulsion components. Systems can include: a substrate including a set of openings; a reservoir facing the substrate at a first side and containing a sample fluid configured for droplet formation upon interacting with the set of openings of the substrate; and a collecting container facing the substrate at a second side and containing a set of fluid layers configured with a density gradient and suitable immiscibility characteristics. One or more components of the system(s) can support methods for emulsion generation, in relation to enabling interactions between multiple continuous phases and a dispersed droplet phase to generate clear emulsions. Applications of the inventions(s) can include performance of droplet-based digital PCR in an improved manner (e.g., without requiring implementation of correction factors based upon Poisson statistics).