NoButter: An R package for reducing transcript dispersion in CosMx Spatial Molecular Imaging Data

Motivation Advances in spatial transcriptomics technologies at single-cell resolution have high-lighted the need for innovative quality assessment approaches and improved analytical tools. Imaging-based spatial transcriptomics technologies, such as the CosMx Spatial Molecular Imager (SMI), provide the location and abundance of transcripts through multifocal imaging. Optical sections (or Z-slices) form a Z-stack that represents the tissue depth. Transcript dispersion can be observed across these Z-slice and introduce considerable levels of technical noise to the data that can negatively impact downstream analysis. Package Functionality NoButter is an R package designed to evaluate transcript dispersion in CosMx SMI spatial transcriptomics data. Using the raw data, the transcript distribution is assessed for each Z-slice of a Z-stack across multiple fields of views (FOVs). To systematically identify transcript dispersion, the percentage of transcripts located outside cell boundaries is calculated. Z-slices exhibiting high levels of transcript dispersion can be excluded, while high-confidence transcripts are preserved. Usage Scenario To demonstrate the functionalities of NoButter, spatial transcriptomics data was generated using the CosMx SMI for lymph node tissue, a lung sample, and two triple-negative breast cancers (TNBCs). Use cases illustrate substantial transcript dispersion in optical planes closer to the glass slide. In these Z-slices, on average, an additional 10% of the transcripts were discarded using NoButter. Cleaning such Z-slices with high dispersion rates reduces technical noise and improves the overall quality of the spatial transcriptomics data. Availability The package can be accessed at https://github.com/cancerbioinformatics/NoButter .