Consecutive hybridization for multiplexed analysis of biological samples

Abstract

Provided herein are high-throughput, high-quality methods of consecutive in situ hybridization for analysis of the genome and/or transcriptome in an individual cell with single-molecule sensitivity. In particular, provided herein are methods comprising visualizing individual genomic loci or transcripts as single detectable signals (e.g., fluorescent spots) which remain in place during consecutive hybridization. In each cycle of consecutive hybridization, detectably labeled probes hybridize to the probe used in the previous cycle, and also introduce the binding sites for the probe of the following cycle. Through consecutive cycles of probe hybridization, imaging, and signal removal, different genomic loci or RNA species can be identified by unique detectable signal profiles (e.g., fluorescent spots with unique color sequences). The number of varied color sequences increases exponentially with the number of hybridization cycles, which enables the genome or transcriptome-wide analysis.