STARTRACER: A COMPUTATIONAL PLATFORM FOR RECONSTRUCTION OF BRAIN-WIDE SYNAPTIC CIRCUITS FROM PARALLELIZED RABIES MONOSYNAPTIC TRACING IN INTACT TISSUE

We introduce StarTracer, a computational platform for high-throughput reconstruction of cell type–resolved synaptic circuits across large volumes of intact brain tissue. StarTracer integrates StarSBARRO, a combinatorial molecular barcoding strategy for parallelized rabies virus–based monosynaptic tracing, with StarMap spatial transcriptomic imaging, enabling simultaneous in situ detection of viral barcodes, host gene expression, and neuronal morphology at cellular resolution in 100-µm brain sections. Using this framework, we demonstrate robust recovery of combinatorial barcodes alongside endogenous mRNAs, reliable identification and morphological imaging of postsynaptic starter cells, and reconstruction of numerous local and long-range monosynaptic networks in mouse brain tissue. StarTracer provides fast, computationally-efficient and user-friendly front and back-end functionality for large-scale StarSBARRO data processing and analysis. Platform functionality includes data ingestion, cell segmentation, barcode decoding, quality control, network reconstruction, graph-based summaries of cell type–resolved monosynaptic connectivity, and interactive visualization for exploratory analysis alongside formal quantification of network properties. The StarTracer package provides a unified platform for principled, multimodal molecular and anatomical mapping of the brain’s cellular and synaptic organization from the micro- to mesoscale and will help facilitate systematic phenotyping of synaptic network properties after experimental manipulations.