Provided herein, in some embodiments, are methods and compositions for highly multiplexed in situ signal amplification via hairpin-mediated concatemerization.

The present invention is directed to methods, compositions and systems for analyzing sequence information while retaining structural and molecular context of that sequence information.

The present invention is directed to methods, compositions and systems for analyzing sequence information while retaining structural and molecular context of that sequence information.

The present invention is directed to methods, compositions and systems for analyzing sequence information while retaining structural and molecular context of that sequence information.

The present invention is directed to methods, compositions and systems for analyzing sequence information while retaining structural and molecular context of that sequence information.

The present disclosure provides methods and assay systems for use in spatially encoded biological assays, including assays to determine a spatial pattern of abundance, expression, and/or activity of one or more biological targets across multiple sites in a sample. In particular, the biological targets comprise proteins, and the methods and assay systems do not depend on imaging techniques for the spatial information of the targets. The present disclosure provides methods and assay systems capable of high levels of multiplexing where reagents are provided to a biological sample in order to address tag the sites to which reagents are delivered; instrumentation capable of controlled delivery of reagents; and a decoding scheme providing a readout that is digital in nature.

The present disclosure provides methods and assay systems for use in spatially encoded biological assays, including assays to determine a spatial pattern of abundance, expression, and/or activity of one or more biological targets across multiple sites in a sample. In particular, the biological targets comprise proteins, and the methods and assay systems do not depend on imaging techniques for the spatial information of the targets. The present disclosure provides methods and assay systems capable of high levels of multiplexing where reagents are provided to a biological sample in order to address tag the sites to which reagents are delivered; instrumentation capable of controlled delivery of reagents; and a decoding scheme providing a readout that is digital in nature.

The present disclosure relates to methods and kits for generating single cell barcodes and imparting them to the constituent molecules within a single cell. Additionally, methods to overlay sample barcode and spatial barcode information onto the single cell barcodes are also described. Generation of single cell barcodes is achieved by labeling the genomic DNA of a cell/nucleus with a small handful, preferably just a one or two cellular barcode probes (CBP) that can be amplified and propagated to label the constituent molecules within the cell. The disclosure finds utility in applications such as characterization of cellular heterogeneity, comprehensive profiling of tissue composition, characterization of adherent cells, discovery of new cell subtypes and functions of individual cells in the context of its microenvironment, and others.

The present disclosure relates to methods and kits for generating single cell barcodes and imparting them to the constituent molecules within a single cell. Additionally, methods to overlay sample barcode and spatial barcode information onto the single cell barcodes are also described. Generation of single cell barcodes is achieved by labeling the genomic DNA of a cell/nucleus with a small handful, preferably just a one or two cellular barcode probes (CBP) that can be amplified and propagated to label the constituent molecules within the cell. The disclosure finds utility in applications such as characterization of cellular heterogeneity, comprehensive profiling of tissue composition, characterization of adherent cells, discovery of new cell subtypes and functions of individual cells in the context of its microenvironment, and others.

Methods of analysis of a sample using hybridization chain reaction (HCR) are provided herein. Some embodiments involve one, two, or all three of the following aspects: 1) repeated signal detection, 2) overlapping binding sites, and 3) catalytic reporter deposition (CARD). Compositions and kits relating to these are also provided. Some embodiments encompass a method for repeated signal detection with reporter-labeled HCR hairpins involving providing a sample possibly containing one or more targets as well as possibly other molecules that are not targets, providing one or more probe sets each comprising either: a) one or more HCR initiator-labeled probes, or b) one or more probe units each comprising two or more HCR fractional initiator probes, providing one or more HCR amplifiers (each labeled with one or more reporters), detecting one or more signals from one or more reporters. In some embodiments, a probe unit comprises two or more HCR fractional initiator probes, wherein an HCR fractional initiator probe comprises a target-binding region and a fractional initiator, wherein the target-binding regions within a probe unit are configured to bind to overlapping or non-overlapping binding sites on the target, and wherein the fractional initiators on the probes within each probe unit are configured to bind to overlapping or non-overlapping binding sites on an HCR hairpin. Some embodiments encompass a method for HCR-mediated catalytic reporter deposition (CARD) for signal detection with hapten-labeled HCR hairpins involving providing a sample possibly containing one or more targets as well as possibly other molecules that are not targets, providing one or more probe sets each comprising either: a) one or more HCR initiator-labeled probes, or b) one or more probe units each comprising two or more HCR fractional initiator probes, providing one or more HCR amplifiers (each labeled with one or more haptens), providing one or more anti-haptens labeled with one or more reporter entities, wherein the reporter entity is an enzyme that mediates CARD, providing one or more CARD-substrates leading to deposition of one or more reporters, and detecting one or more signals from one or more reporters.