Abstract
Dual RNA-sequencing enables simultaneous profiling of protein-coding and non-coding transcripts from two interacting organisms, an essential capability when physical separation is difficult, such as in host-parasite or cross-kingdom interactions (e.g., plant-plant or host-pathogen systems). By allowing in silico separation of mixed reads, dual RNA-seq reveals the transcriptomic dynamics of both partners during interaction. However, existing analysis workflows often require programming expertise, limiting accessibility. We present inDAGO, a free, open-source, cross-platform graphical user interface designed for biologists without coding skills. inDAGO supports both bulk and dual RNA sequencing, with dual RNA sequencing further accommodating both sequential and combined approaches. The interface guides users through key analysis steps, including quality control, read alignment, read summarization, exploratory data analysis, and identification of differentially expressed genes, while generating intermediate outputs and publication-ready plots. Optimized for speed and efficiency, inDAGO performs complete analyses on a standard laptop (16 GB RAM) without requiring high-performance computing. We validated inDAGO using diverse real datasets to demonstrate its reliability and usability. inDAGO, available on CRAN (https://cran.r-project.org/web/packages/inDAGO/) and GitHub (https://github.com/inDAGOverse/inDAGO), lowers the technical barrier to dual RNA-seq by enabling robust, reproducible analyses, even for users without coding experience.