BILBO: a self-hosted platform for large-scale RNA-Seq analysis with a functional annotation clustering framework

RNA sequencing (RNA-Seq) has become a central approach for transcriptomic studies, but its practical application still requires the coordination of multiple computational steps and often produces large gene expression outputs whose integrative biological interpretation remains challenging. Here, we present BILBO (Bioinformatics Integration for Large-scale Biological Operations), a self-hosted and fully containerized platform designed to support reproducible, end-to-end RNA-Seq analyses, from data acquisition to integrative functional interpretation. BILBO consolidates widely adopted open-source tools into standardized workflows, enabling automated retrieval of public or private RNA-Seq data, quality control, alignment, quantification, normalization, differential expression analysis, and generation of publication-ready outputs within a unified environment. Beyond workflow integration, BILBO introduces a novel functional annotation clustering framework that organizes genes using Gene Ontology (GO)-based semantic similarity. The approach separates genes by regulatory direction and ontology, computes Wang semantic similarity among GO annotations, applies complete-linkage hierarchical clustering, refines assignments through silhouette-based pruning, and selects representative genes as semantic medoids. To further enhance biological interpretability, BILBO integrates a Retrieval-Augmented Generation strategy that links representative genes and functional clusters to curated scientific literature through hybrid retrieval and DOI-linked evidence chunks. In a controlled SEQC/MAQC-III benchmark against nf-core/rnaseq, BILBO preserved differential-expression effect sizes (cross-workflow Spearman ≥ 0.994) and the expected behavior of the reference mixtures, whereas BILBO required 68% less peak temporary disk space but 25% more wall time. In the SEQC dataset, the resulting clusters showed strong coherence with independent MSigDB pathways, comparable to a GOSemSim-based reference workflow, and remained stable under moderate parameter changes. Together, these features position BILBO as an integrative and scalable solution that bridges the gap between large-scale RNA-Seq data processing and higher-level functional insight, promoting reproducibility, interpretability, and accessibility in transcriptomic research.