High-throughput sequencing technologies have advanced RNA virus genomics, but recovering viral genomes from mammalian tissues remains challenging due to the predominance of host RNA. We evaluated two metatranscriptomic workflows to address these challenges. Our results demonstrate that the methods differed significantly in performance, with Method B achieving a 5-fold increase in RNA yield and improved RNA integrity over Method A. These differences resulted in the recovery of 4 complete hepacivirus genomes with Method B compared to fragmented or incomplete genomes with Method A. Additionally, Method B's library preparation workflow, incorporating rRNA depletion, enhanced viral genome recovery by reducing host RNA background. Our novel approach integrates an optimized RNA purification protocol with a customized bioinformatics strategy for improved viral genome recovery. Overall, our findings highlight the critical role of optimized homogenization, RNA purification, and library preparation in metatranscriptomic workflows, facilitating the more effective RNA virus genome recovery from complex mammalian tissues.

Bioinformatics and Applied Genomics Department of Microbiology Hellenic Pasteur Institute Athens Greece

Department of Hygiene Epidemiology and Medical Statistics School of Medicine National and Kapodistrian University of Athens Athens Greece

Department of Microbiology Immunology and Transplantation KU Leuven Rega Institute KU Leuven Leuven Belgium

EcoHealth Alliance New York New York United States of America

Evolutionary Ecology group Department of Biology University of Antwerp Antwerp Belgium

Institute of Vertebrate Biology The Czech Academy of Sciences Brno Czech Republic

Ruijin Hospital Shanghai Jiao Tong University School of Medicine Shanghai PR China

Spatial Epidemiology Lab Université Libre de Bruxelles Bruxelles Belgium

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