The present disclosure relates in some aspects to methods for analyzing a target nucleic acid in a biological sample. In some aspects, provided herein are methods and compositions for detecting a region of interest in a target nucleic acid.

The present disclosure relates in some aspects to methods for analyzing a target nucleic acid in a biological sample. In some aspects, provided herein are methods and compositions for detecting a region of interest in a target nucleic acid.

The present disclosure relates in some aspects to methods and compositions for analysis of a target nucleic acid, such as in situ detection of a region of interest in a polynucleotide in a tissue sample. In some embodiments, provided herein are templated ligation probes (e.g., RNA-templated ligation probes) and selector probes for generation of a circularized ligated probe comprising an insertion sequence of a selector probe, wherein the circularized ligated probe is amplified in a rolling circle amplification reaction to generate a product that is detected in the sample.

The present disclosure relates in some aspects to methods and compositions for analysis of a target nucleic acid, such as in situ detection of a region of interest in a polynucleotide in a tissue sample. In some embodiments, provided herein are templated ligation probes (e.g., RNA-templated ligation probes) and selector probes for generation of a circularized ligated probe comprising an insertion sequence of a selector probe, wherein the circularized ligated probe is amplified in a rolling circle amplification reaction to generate a product that is detected in the sample.

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 allow for detection of a target sequence by rolling circle amplification without requiring a ligation step and without sacrificing specificity (e.g., rolling circle amplification occurs only for circular probes and/or hairpin molecules specifically hybridized to a target sequence).

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 allow for detection of a target sequence by rolling circle amplification without requiring a ligation step and without sacrificing specificity (e.g., rolling circle amplification occurs only for circular probes and/or hairpin molecules specifically hybridized to a target sequence).

The present invention provides a method for improving the loading of nucleic acid on a solid support by contacting the solid support with a poloxamer-containing reagent. The present invention also provides a method for improving the stability of a nucleic acid on a solid support, comprising contacting a nucleic acid molecule with a partially double-strand oligonucleotide before or after loading the nucleic acid molecule on a solid support, so as to cause the nucleic acid molecule to hybridize with the oligonucleotide. The present invention also provides a combined use of the two methods.

The present invention provides a method for improving the loading of nucleic acid on a solid support by contacting the solid support with a poloxamer-containing reagent. The present invention also provides a method for improving the stability of a nucleic acid on a solid support, comprising contacting a nucleic acid molecule with a partially double-strand oligonucleotide before or after loading the nucleic acid molecule on a solid support, so as to cause the nucleic acid molecule to hybridize with the oligonucleotide. The present invention also provides a combined use of the two methods.

A method for determining sequences from sense and antisense strands of a nucleic acid, including (a) providing a nucleic acid cluster attached to a solid support, wherein the nucleic acid cluster includes a sense strand and an antisense strand of a concatemer, the concatemer including multiple copies of a sequence unit, the sequence unit including a target sequence and a primer binding site; (b) hybridizing a primer to a primer binding site in the antisense strand; (c) extending the primer along the antisense strand to determine the sequence from at least a portion of the target sequence in the antisense strand; (d) hybridizing a second primer to a primer binding site in the sense strand; and (e) extending the second primer along the sense strand to determine the sequence from at least a portion of the target sequence in the sense strand.

A method for determining sequences from sense and antisense strands of a nucleic acid, including (a) providing a nucleic acid cluster attached to a solid support, wherein the nucleic acid cluster includes a sense strand and an antisense strand of a concatemer, the concatemer including multiple copies of a sequence unit, the sequence unit including a target sequence and a primer binding site; (b) hybridizing a primer to a primer binding site in the antisense strand; (c) extending the primer along the antisense strand to determine the sequence from at least a portion of the target sequence in the antisense strand; (d) hybridizing a second primer to a primer binding site in the sense strand; and (e) extending the second primer along the sense strand to determine the sequence from at least a portion of the target sequence in the sense strand.