Barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel in situ analyses

Abstract

We present barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel insitu analyses (BOLORAMIS), a reverse transcription-free method for spatially-resolved, targeted, in situ RNA identification of single or multiple targets. BOLORAMIS was demonstrated on a range of cell types and human cerebral organoids. Singleplex experiments to detect coding and non-coding RNAs in human iPSCs showed a stem-cell signature pattern. Specificity of BOLORAMIS was found to be 92% as illustrated by a clear distinction between human and mouse housekeeping genes in a co-culture system, as well as by recapitulation of subcellular localization of lncRNA MALAT1. Sensitivity of BOLORAMIS was quantified by comparing with single molecule FISH experiments and found to be 11%, 12% and 35% for GAPDH, TFRC and POLR2A, respectively. To demonstrate BOLORAMIS for multiplexed gene analysis, we targeted 96 mRNAs within a co-culture of iNGN neurons and HMC3 human microglial cells. We used fluorescence in situ sequencing to detect error-robust 8-base barcodes associated with each of these genes. We then used this data to uncover the spatial relationship among cells and transcripts by performing single-cell clustering and gene–gene proximity analyses. We anticipate the BOLORAMIS technology for in situ RNA detection to find applications in basic and translational research.

Museum of spatial transcriptomics

“…RCA is commonly used for signal amplification. The 2013 ISS 59 , later commercialized by Cartana, and barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel in situ analyses (BOLORAMIS) 60 use one query base per probe, as in combinatorial probe anchor ligation (cPAL) 61 , to sequence gene barcodes (Fig. 2e).…”

Advances and Challenges in Fluorescence in situ Hybridization for Visualizing Fungal Endobacteria

“…Small subunit (SSU) rRNA sequences for both the fungal host (NCBI accession ON357719) and the bacterial endosymbiont (ON357872) were extracted for use in probe design. Taxonomic-specific FISH probes were designed using Python code BioPython package adapted from Liu et al (2021) . In summary, the script was written for the specific purpose of parsing both the 16S rRNA sequence from the bacterial endosymbiont and the 18S rRNA sequence from the fungal host.…”

The Known Unknowns of the Immune Response to Coccidioides

“…However, detection efficiency and sensitivity were low and thus, could be problematic in tissue when detecting low levels of RNA targets in certain disease states. Recently, the group that developed FISSEQ optimized this approach by directly targeting mRNA, resulting in higher efficiency and lower background, thereby increasing signal-to-noise [ 134 ], used padlock probes specifically designed for target mRNA, amplified the circularized proves, and sequenced these barcodes over multiple rounds. BOLORAMIS allows for visualization of transcripts and uncovers the spatial relationship between cells and transcripts via gene-gene proximity and single-cell clustering analyses.…”