In some aspects, the present disclosure relates to methods for reducing the crowding of signals, for example optical crowding, that can occur when nucleic acids are detected in a sample in multiplex, which can make it difficult to resolve individual signals and can lead to a reduced dynamic range. In some aspects, the present disclosure relates to methods for reducing signal crowding in the detection of multiple target nucleic acid sequences in a sample, e.g., using hybridization probes, wherein signal crowding from said hybridization probes is reduced. The methods herein have particular applicability in the detection of barcode sequences by sequencing-by-hybridization (SBH) methods, including those relying on combinatorial labelling schemes and decoding of the barcodes by sequential cycles of decoding using hybridization probes. Also provided are kits comprising probes for use in such methods.

The present invention is directed to improved methods for conducting immunoassays. The methods are designed to amplify signals in immunoassays and anchor immunoassay complexes employed therein.

The present invention is directed to improved methods for conducting immunoassays. The methods are designed to amplify signals in immunoassays and anchor immunoassay complexes employed therein.

The invention is directed to a method to obtain the spatial location and sequence information of at least a part of a RNA or cDNA strand (006) in a sample comprising the steps a. hybridizing a first detection probe oligonucleotide (204) comprising 50-1000 nucleotides with its 3′ and/or 5′ end to the complementary part of the at least one RNA or cDNA strand, wherein the detection probe oligonucleotide is partially hybridized to a bridge oligonucleotide (205) comprising 5-100 nucleotides wherein a gap region (206) capable of binding oligonucleotides is created b. filling the gap region (206) in part with 1 to 16 barcode oligonucleotides comprising 4-20 nucleotides, wherein the barcode oligonucleotides determine the spatial information of the RNA or cDNA strand in the sample c. partially hybridizing a second detection probe oligonucleotide (204′) comprising 50-1000 nucleotides with its 3′ and/or 5′ end to the complementary part of the same or cDNA strand and with the respective other end to the bridge oligonucleotide (205) to create a circular template d. multiplying the circular template by a polymerase capable of rolling circle amplification into rolonies comprising a plurality of concatemers e. determining the sequence of nucleotides of the rolonies

The invention is directed to a method to obtain the spatial location and sequence information of at least a part of a RNA or cDNA strand (006) in a sample comprising the steps a. hybridizing a first detection probe oligonucleotide (204) comprising 50-1000 nucleotides with its 3′ and/or 5′ end to the complementary part of the at least one RNA or cDNA strand, wherein the detection probe oligonucleotide is partially hybridized to a bridge oligonucleotide (205) comprising 5-100 nucleotides wherein a gap region (206) capable of binding oligonucleotides is created b. filling the gap region (206) in part with 1 to 16 barcode oligonucleotides comprising 4-20 nucleotides, wherein the barcode oligonucleotides determine the spatial information of the RNA or cDNA strand in the sample c. partially hybridizing a second detection probe oligonucleotide (204′) comprising 50-1000 nucleotides with its 3′ and/or 5′ end to the complementary part of the same or cDNA strand and with the respective other end to the bridge oligonucleotide (205) to create a circular template d. multiplying the circular template by a polymerase capable of rolling circle amplification into rolonies comprising a plurality of concatemers e. determining the sequence of nucleotides of the rolonies

Aspects of the present disclosure include methods of making barcoded solid supports. In some embodiments, the methods include producing a concatemer by rolling circle amplification (RCA) of a circular nucleic acid template, where the circular nucleic acid template includes a barcode and a stem-loop forming region, and where the concatemer includes a plurality of linked units, each unit including the barcode and a stem-loop structure formed from the stem-loop forming region. Such methods further include disposing the concatemer on a solid support to produce a barcoded solid support including a plurality of the stem-loop structures extending from the surface of the solid support. The methods may further include treating the stem-loop structures with an agent that produces stem structures having ends compatible with target nucleic acids, and attaching the target nucleic acids to the stem structures. Barcoded solid supports and methods of using the barcoded solid supports are also provided.

Aspects of the present disclosure include methods of making barcoded solid supports. In some embodiments, the methods include producing a concatemer by rolling circle amplification (RCA) of a circular nucleic acid template, where the circular nucleic acid template includes a barcode and a stem-loop forming region, and where the concatemer includes a plurality of linked units, each unit including the barcode and a stem-loop structure formed from the stem-loop forming region. Such methods further include disposing the concatemer on a solid support to produce a barcoded solid support including a plurality of the stem-loop structures extending from the surface of the solid support. The methods may further include treating the stem-loop structures with an agent that produces stem structures having ends compatible with target nucleic acids, and attaching the target nucleic acids to the stem structures. Barcoded solid supports and methods of using the barcoded solid supports are also provided.

The invention is directed to a method to obtain the spatial location and sequence information of a target sequence in a sample comprising at least one m-RNA strand comprising the steps a. providing a surface with a plurality of spacer units capable of binding at least one m-RNA strand and with at least one fiducial marker b. providing a sample comprising at least one m-RNA strand to the surface wherein at least one m-RNA strand of the sample binds to at least one spacer unit creating at least one single stranded oligomer c. taking a first image of the surface to obtain the spatial information of the sample relative to the fiducial marker d. removing sample form surface e. hybridizing at least one oligonucleotide comprising 50-1000 nucleic acids with its 5′ and 3′ ends to complementary parts of the single stranded oligomer thereby forming a padlock-shaped structure that is ligated to create a single strand circular template f. multiplying the single strand circular template by a polymerase capable of rolling circle amplification into a plurality of DNA concatemers thereby forming rolonies g. obtaining the sequence information of the rolonies h. linking the spatial information of the sample with the sequence information of the rolonies.

The invention is directed to a method to obtain the spatial location and sequence information of a target sequence in a sample comprising at least one m-RNA strand comprising the steps a. providing a surface with a plurality of spacer units capable of binding at least one m-RNA strand and with at least one fiducial marker b. providing a sample comprising at least one m-RNA strand to the surface wherein at least one m-RNA strand of the sample binds to at least one spacer unit creating at least one single stranded oligomer c. taking a first image of the surface to obtain the spatial information of the sample relative to the fiducial marker d. removing sample form surface e. hybridizing at least one oligonucleotide comprising 50-1000 nucleic acids with its 5′ and 3′ ends to complementary parts of the single stranded oligomer thereby forming a padlock-shaped structure that is ligated to create a single strand circular template f. multiplying the single strand circular template by a polymerase capable of rolling circle amplification into a plurality of DNA concatemers thereby forming rolonies g. obtaining the sequence information of the rolonies h. linking the spatial information of the sample with the sequence information of the rolonies.

The present disclosure relates in some aspects to methods, probes, kits, and compositions for analysis of a target nucleic acid, such as in situ generation of a circular probe for detection of a target nucleic acid in a tissue sample.