Highly multiplexed spatial mapping of transcripts within tissues allows for investigation of the transcriptomic and cellular diversity of mammalian organs previously unseen. Here we explore a direct RNA (dRNA) detection approach incorporating the use of padlock probes and rolling circle amplification in combination with hybridization-based in situ sequencing chemistry. We benchmark a High Sensitivity Library Preparation Kit from CARTANA that circumvents the reverse transcription needed for cDNA-based in situ sequencing (ISS) via direct RNA detection. We found a fivefold increase in transcript detection efficiency when compared to cDNA-based ISS and also validated its multiplexing capability by targeting a curated panel of 50 genes from previous publications on mouse brain sections, leading to additional data interpretation such as de novo cell clustering. With this increased efficiency, we also found to maintain specificity, multiplexing capabilities and ease of implementation. Overall, the dRNA chemistry shows significant improvements in target detection efficiency, closing the gap to other fluorescent in situ hybridization-based technologies and opens up possibilities to explore new biological questions previously not possible with cDNA-based ISS.

Multiplexed spatial profiling of mRNAs has recently gained traction as a tool to explore the cellular diversity and the architecture of tissues. We propose a sensitive, open-source, simple and flexible method for the generation of in situ expression maps of hundreds of genes. We use direct ligation of padlock probes on mRNAs, coupled with rolling circle amplification and hybridization-based in situ combinatorial barcoding, to achieve high detection efficiency, high-throughput and large multiplexing. We validate the method across a number of species and show its use in combination with orthogonal methods such as antibody staining, highlighting its potential value for developmental and tissue biology studies. Finally, we provide an end-to-end computational workflow that covers the steps of probe design, image processing, data extraction, cell segmentation, clustering and annotation of cell types. By enabling easier access to high-throughput spatially resolved transcriptomics, we hope to encourage a diversity of applications and the exploration of a wide range of biological questions.

In-situ detection of expressed point mutations within fixed cells or tissues is challenging due to the low sensitivity or poor specificity of available molecular tools. Here, we present KODFISH, an efficient and specific approach based on single nucleotide sensitive RNA-based in-situ sequencing (RNA-ISS) using padlock probes and rolling circle amplification. KODFISH enables the accurate in-situ discrimination of single nucleotide changes, allowing the spatial detection of expressed mutations with higher sensitivity and specificity than currently available methods.

Detecting complex mutations in-situ within fixed cells or tissues is challenging, because of low sensitivity or poor specificity. We developed an efficient and specific molecular tool based on the RNA-templated and controlled gap-filling of padlock probes. Our method enables the accurate in-situ sequencing of unknown RNA stretches surrounded by known flanking regions, allowing the in-situ detection of expressed mutations with higher sensitivity and specificity than currently available tools.

Schizophrenia is a heritable and genetically complex disorder with poorly understood aetiology. Previous study from our lab has shown that this disorder could affect a limited number of cell types in the brain1. Here, we build on these findings and present the first spatially-resolved transcriptomics study of schizophrenia with a large number of samples (17 in total). By comparing spatial cell type distributions in diseased and healthy prefrontal cortices, we discover that certain non-neuronal cell types change their co-localization patterns