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.

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.

There is provided a method of detecting the presence of a nucleic acid target sequence in which two oligonucleotides are used to forma three-way junction with the target sequence to allow detection of the target sequence. Alternatively, three oligonucleotides can be used to form a four-way junction with the target sequence to allow detection of the target sequence.

There is provided a method of detecting the presence of a nucleic acid target sequence in which two oligonucleotides are used to forma three-way junction with the target sequence to allow detection of the target sequence. Alternatively, three oligonucleotides can be used to form a four-way junction with the target sequence to allow detection of the target sequence.

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.

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.

Provided herein is a circular proximity ligation assay in which proximity-probes are employed as bridges to connect two free oligonucleotides via a dual ligation event, resulting in the formation of a circle. The circles are then quantified by, e.g., qPCR. The addition of an extra oligonucleotide is believed to enhance specificity by decreasing the probability of random background ligation events. In addition, circle formation may have selective advantages, as uncircularized DNA can be removed by a simple exonuclease treatment and it has streamlined the workflow by eliminating preamplification prior to qPCR.

Provided herein is a circular proximity ligation assay in which proximity-probes are employed as bridges to connect two free oligonucleotides via a dual ligation event, resulting in the formation of a circle. The circles are then quantified by, e.g., qPCR. The addition of an extra oligonucleotide is believed to enhance specificity by decreasing the probability of random background ligation events. In addition, circle formation may have selective advantages, as uncircularized DNA can be removed by a simple exonuclease treatment and it has streamlined the workflow by eliminating preamplification prior to qPCR.

Provided are methods and kits for detecting and quantifying a sequence difference relative to a target polynucleotide sequence, and methods for developing assays. Target polynucleotide sequences are examined to determine if any of the sequences vary by at least one nucleotide difference. Promiscuous probes that have on and off-target binding at different binding efficiencies at permissive temperature are used, so that differences in polynucleotide sequences are reliably detected and quantified in a single well with fewer types of labeled probes, including by polymerase chain reaction of any of a range of sequences where there is interest in detecting an at least one nucleotide difference relative. The target sequences may be a reference polynucleotide sequence indicative of a first state, such as “normal” and another sequence that varies by at least one polynucleotide indicative of a different second state, such as a mutation, disease condition, or predisposition thereto.

Provided are methods and kits for detecting and quantifying a sequence difference relative to a target polynucleotide sequence, and methods for developing assays. Target polynucleotide sequences are examined to determine if any of the sequences vary by at least one nucleotide difference. Promiscuous probes that have on and off-target binding at different binding efficiencies at permissive temperature are used, so that differences in polynucleotide sequences are reliably detected and quantified in a single well with fewer types of labeled probes, including by polymerase chain reaction of any of a range of sequences where there is interest in detecting an at least one nucleotide difference relative. The target sequences may be a reference polynucleotide sequence indicative of a first state, such as “normal” and another sequence that varies by at least one polynucleotide indicative of a different second state, such as a mutation, disease condition, or predisposition thereto.