TrAnnoScope: A Modular Snakemake Pipeline for Full-Length Transcriptome Analysis and Functional Annotation

Background/Objectives: Transcriptome assembly and functional annotation are essential in understanding gene expression and biological function. Nevertheless, many existing pipelines lack the flexibility to integrate both short- and long-read sequencing data or fail to provide a complete, customizable workflow for transcriptome analysis, particularly for non-model organisms. Methods: We present TrAnnoScope, a transcriptome analysis pipeline designed to process Illumina short-read and PacBio long-read data. The pipeline provides a complete, customizable workflow to generate high-quality, full-length (FL) transcripts with broad functional annotation. Its modular design allows users to adapt specific analysis steps for other sequencing platforms or data types. The pipeline encompasses steps from quality control to functional annotation, employing tools and established databases such as SwissProt, Pfam, Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), and Eukaryotic Orthologous Groups (KOG). As a case study, TrAnnoScope was applied to RNA-Seq and Iso-Seq data from zebra finch brain, ovary, and testis tissue. Results: The zebra finch transcriptome generated by TrAnnoScope from the brain, ovary, and testis tissue demonstrated strong alignment with the reference genome (99.63%), and it was found that 93.95% of the matched protein sequences in the zebra finch proteome were captured as nearly complete. Functional annotation provided matches to known protein databases and assigned relevant functional terms to the majority of the transcripts. Conclusions: TrAnnoScope successfully integrates short and long sequencing technologies to generate transcriptomes with minimal user input. Its modularity and ease of use make it a valuable tool for researchers analyzing complex datasets, particularly for non-model organisms.