Provided herein are methods of proximity ligation and compositions for use in such methods. Also provided herein are embodiments related to single cell nucleic acid conformation assessment or single cell nucleic acid sequence or phase information determination or conformation-reconstructed nucleic acid samples can be fragmented and distributed in aliquots to which aliquot-distinguishing sequence segments are added so that, upon analysis of a paired end library generated from the samples, paired ends are assigned to a partition, or cell, of origin. Thus cell-specific variation in sequence and three-dimensional nucleic acid configuration can be determined.

Provided herein are methods of proximity ligation and compositions for use in such methods. Also provided herein are embodiments related to single cell nucleic acid conformation assessment or single cell nucleic acid sequence or phase information determination or conformation-reconstructed nucleic acid samples can be fragmented and distributed in aliquots to which aliquot-distinguishing sequence segments are added so that, upon analysis of a paired end library generated from the samples, paired ends are assigned to a partition, or cell, of origin. Thus cell-specific variation in sequence and three-dimensional nucleic acid configuration can be determined.

Labeled probes, and methods of use thereof, comprise a Cas polypeptide conjugated to gRNA that is specific for target nucleic acid sequences, including genomic DNA sequences. The probes and methods can be used to label nucleic acid sequences without global DNA denaturation. The presently-disclosed subject matter meets some or all of the above identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.

Labeled probes, and methods of use thereof, comprise a Cas polypeptide conjugated to gRNA that is specific for target nucleic acid sequences, including genomic DNA sequences. The probes and methods can be used to label nucleic acid sequences without global DNA denaturation. The presently-disclosed subject matter meets some or all of the above identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.

A method for determining the presence or absence of variant ribonucleic acid molecules in a population of ribonucleic acid molecules, wherein the reference sequence of the variant ribonucleic acid molecules is known, the method comprising: (a) interrogating the population of ribonucleic acid molecules with a plurality of primer molecules and a plurality of probes so as to saturate the population of ribonucleic acid molecules with the probes and primer molecules such that the probes and primer molecules are adjacent to one another when hybridized to their respective complimentary sequences on the ribonucleic acid molecules, (b) ligating the probes to their respective adjacent primer molecules so as to form ligated nucleic acid molecules, and (c) detecting the presence of ligated nucleic acid molecules that are complementary to a sequence that differs from the reference sequence, thereby determining the presence or absence of one or more variant ribonucleic acid molecules in the population of ribonucleic acid molecules.

A method for determining the presence or absence of variant ribonucleic acid molecules in a population of ribonucleic acid molecules, wherein the reference sequence of the variant ribonucleic acid molecules is known, the method comprising: (a) interrogating the population of ribonucleic acid molecules with a plurality of primer molecules and a plurality of probes so as to saturate the population of ribonucleic acid molecules with the probes and primer molecules such that the probes and primer molecules are adjacent to one another when hybridized to their respective complimentary sequences on the ribonucleic acid molecules, (b) ligating the probes to their respective adjacent primer molecules so as to form ligated nucleic acid molecules, and (c) detecting the presence of ligated nucleic acid molecules that are complementary to a sequence that differs from the reference sequence, thereby determining the presence or absence of one or more variant ribonucleic acid molecules in the population of ribonucleic acid molecules.

The present invention provides assays and assay systems for use in spatially encoded biological assays. The invention provides an assay system comprising an assay capable of high levels of multiplexing where reagents are provided to a biological sample in defined spatial patterns; instrumentation capable of controlled delivery of reagents according to the spatial patterns; and a decoding scheme providing a readout that is digital in nature.

The present invention provides assays and assay systems for use in spatially encoded biological assays. The invention provides an assay system comprising an assay capable of high levels of multiplexing where reagents are provided to a biological sample in defined spatial patterns; instrumentation capable of controlled delivery of reagents according to the spatial patterns; and a decoding scheme providing a readout that is digital in nature.

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.

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.