OligoY pipeline for full Y chromosome painting.

The Y chromosome's unique structure poses challenges for cytogenetic studies, especially in designing probes for FISH Oligopaint labeling experiments (Fluorescence in situ hybridization). The standard protocol for designing probes for these experiments discards repetitive sequences to avoid off-target hybridization. Given the highly repetitive nature of the Y chromosome, assemblies often remain fragmented, leaving significant regions incompletely sequenced, unprobed, and poorly understood. Among these, the remaining nonrepetitive sequences are usually insufficient to design probes and efficiently perform FISH Oligopaint assays, since they do not cover most regions of the chromosome. This limitation hinders comprehensive cytogenetic studies, which are crucial not only for understanding the Y chromosome's role in genetics but also for broader applications in evolutionary biology, medicine, and conservation. Here, we introduce a new computational pipeline to design full chromosome fluorescent labeling probes for the Y chromosome of any species of interest. Based on open-source tools, the OligoY pipeline increases the amount of contigs assigned to the Y chromosome from the reference genome assembly, and effectively uses repetitive sequences unique to the target chromosome to design probes. Throughout its steps, the pipeline gives the user the autonomy to choose parameters, maximizing the overall efficiency of cytogenetic experiments. After extensive in silico and in situ testing and validation with the human and Drosophila melanogaster genomes, we show for the first time a pipeline for FISH Oligopaint probe design that significantly increases previous Y chromosome staining with no off-target signal.