Abstract: The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying multiple analytes present in a biological sample. In some aspects, the methods and compositions provided herein address one or more issues associated with the stability and/or size of nucleic acid structures, such as RCPs, in the biological sample without the use of exogenously added oligonucleotide compaction probes. In some embodiments, provided herein are methods involving the use of self-hybridizing hybridizing regions for compacting and/or stabilizing nucleic acid concatemers (e.g., RCPs). In some embodiments, dynamic inter-strand annealing between tandem units of an RCP is used for compaction and/or stabilization. In some embodiments, short palindromic regions in an RCP are used for compaction and/or stabilization.

Abstract: 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.

Abstract: The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying multiple analytes present in a biological sample. In some aspects, the methods and compositions provided herein address one or more issues associated with the stability and/or size of nucleic acid structures, such as RCPs, in the biological sample without the use of exogenously added oligonucleotide compaction probes. In some embodiments, provided herein are methods involving the use of self-hybridizing hybridizing regions for compacting and/or stabilizing nucleic acid concatemers (e.g., RCPs). In some embodiments, dynamic inter-strand annealing between tandem units of an RCP is used for compaction and/or stabilization. In some embodiments, short palindromic regions in an RCP are used for compaction and/or stabilization.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: Methods for index hopping sequence read filtering are provided. Each read in a plurality of reads from a multiplexed reaction comprises an insert portion, and first (molecular identifier) and second (sample index) non-insert portions. For each of a plurality of hashes, a hash data structure is formed with a representation of each read. Each representation comprises a hash of the first non-insert portion of the corresponding read. Read pairs are identified in the hash data structures. Each pair includes a first and second read sharing a common hash value but differing index values. An entry is added into a heterogeneous data structure, for each such pair, that includes the first and second non-insert portions of the first and second reads of the pair. Reads with first non-insert portion values appearing more than a threshold number of times in the heterogeneous data structure are removed from the plurality of reads.

Abstract: The present disclosure relates in some aspects to methods and compositions for assessing performance of in situ analyte detection and/or platforms (e.g., methods, assays, workflows, and/or instruments for in situ analyte detection and/or sequencing). In some aspects, performance can be assessed for an individual platform, or the performance of two or more different platforms can be compared. In some aspects, assessing performance comprises a standardized in situ analyte detection workflow and analysis on a test biological sample comprising defined cell populations.

Abstract: Methods and compositions for performing base-by-base sequencing in situ in a cell or tissue sample that minimize optical crowding are described. In some embodiments, a sequencing primer hybridizes to a priming site 3? to an identifier sequence (e.g., a barcode sequence) in the sample such that the sequencing primer can be extended by a polymerase in a base-by-base fashion using the identifier sequence as a template. The sample can be contacted with nucleotides in a cyclic series of nucleotide incorporation or binding steps, and signals indicative of the incorporation or binding events can be detected to generate signal code sequences comprising a series of signal codes (corresponding to signals (ON signals), absence of signals (OFF signals), or a combination thereof) detected in the sequential cycles. Decoding of the identifier sequences based at least in part of the signal code sequences can be used to detect and locate the corresponding analytes.

Abstract: The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying multiple analytes present in a biological sample. In some aspects, the methods and compositions provided herein address one or more issues associated with the stability and/or size of nucleic acid structures, such as RCPs, in the biological sample without the use of exogenously added oligonucleotide compaction probes. In some embodiments, provided herein are methods involving the use of self-hybridizing hybridizing regions for compacting and/or stabilizing nucleic acid concatemers (e.g., RCPs). In some embodiments, dynamic inter-strand annealing between tandem units of an RCP is used for compaction and/or stabilization. In some embodiments, short palindromic regions in an RCP are used for compaction and/or stabilization.

Abstract: 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.

Abstract: Methods for index hopping sequence read filtering are provided. Each read in a plurality of reads from a multiplexed reaction comprises an insert portion, and first (molecular identifier) and second (sample index) non-insert portions. For each of a plurality of hashes, a hash data structure is formed with a representation of each read. Each representation comprises a hash of the first non-insert portion of the corresponding read. Read pairs are identified in the hash data structures. Each pair includes a first and second read sharing a common hash value but differing index values. An entry is added into a heterogeneous data structure, for each such pair, that includes the first and second non-insert portions of the first and second reads of the pair. Reads with first non-insert portion values appearing more than a threshold number of times in the heterogeneous data structure are removed from the plurality of reads.