On-site microbial community analysis in rivers: integrating autonomous sampling with a portable sequencing workflow

Studies focused on bacterial diversity in rivers often face significant limitations. Traditional sampling approaches frequently overlook spatial and temporal variability at the reach scale. Molecular techniques, such as metabarcoding and metagenomics, can partially address this issue by providing deeper insights. However, this approach usually involves highly specialized laboratories and high costs, making large-scale and long-term monitoring of river networks unfeasible in most cases. Thus, we developed a methodology based on remote-controlled boats, allowing the collection of integrated samples in freshwater ecosystems. Combined with a portable laboratory, this approach enabled the development of new surface water monitoring strategies. Here, we describe the operational application of this system for in situ monitoring of bacterioplankton communities across 8 sections of the Ter River (Catalonia, Spain). [To explore its potential, we applied both 16S rRNA gene sequencing using Nanopore technology (MinION) in the field, and shotgun metagenomics using Illumina technology in the laboratory, acknowledging the intrinsic differences between sequencing targets, platforms, and analysis pipelinesMinIONMinION sequencing enabled microbiome characterization and identification of the main bacterial taxa just 72 h after sampling, offering significantly lower costs and reduced manpower requirements. Furthermore, amplification facilitated the full characterization of bacterioplankton diversity along the river, preventing the exclusion of uncommon taxa. While shotgun metagenomics is still necessary for understanding the functional activity of these organisms, our approach provides a cost-effective framework for developing efficient follow-up sampling methodologies.