Nucleic acid assays using click chemistry bioconjugation

Abstract

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.