A unified pipeline for FISH spatial transcriptomics

High-throughput spatial transcriptomics has emerged as a powerful tool for investigating the spatial distribution of mRNA expression and its effects on cellular function. There is a lack of standardized tools for analyzing spatial transcriptomics data, leading many groups to write their own in-house tools that are often poorly documented and not generalizable. To address this, we have expanded and improved the starfish library and used those tools to create PIPEFISH, a semi-automated and generalizable pipeline that performs transcript annotation for fluorescence in situ hybridization (FISH)-based spatial transcriptomics. We used this pipeline to annotate transcript locations from three real datasets from three different common types of FISH image-based experiments, MERFISH, seqFISH, and targeted in situ sequencing (ISS), and verified that the results were high quality using the internal quality metrics of the pipeline and also a comparison with an orthogonal method of measuring RNA expression. PIPEFISH is a publicly available and open-source tool.