A FAIR Amplicon Sequencing Workflow for Long-term Environmental Monitoring

Antarctica represents one of the last pristine environments on Earth, providing a unique opportunity to to study the effects of climate change and anthropogenic activities. Ice-free areas, such as the inland nunataks of the Sor Rondane Mountains (SRM), host unique terrestrial and lacustrine ecosystems, of which the simplified food webs rely almost exclusively on microbial primary production. Because of their small size, low productivity and hence low biomass, these microbial communities are fragile. Seven SRM sites were selected to be part of the Antarctic Specially Protected Area (ASPA) 179. The MonASPA project has established an environmental monitoring program to evaluate the effectiveness of the management plan approved by the Antarctic Treaty System for these areas. A key component of MonASPA is the long-term monitoring of microbial biodiversity using 16S rRNA amplicon sequencing. To ensure consistent operation over decades, we developed the Reproducible Amplicon Sequencing Pipeline for Antarctic Monitoring (RASPAM), which is built on Apptainer containers and Nextflow workflows. RASPAM implements Amplicon Sequence Variants (ASVs) and Zero-radius Operational Taxonomic Units (ZOTUs) to provide high-resolution taxonomic affiliation. It incorporates, in addition to the SILVA database, a taxonomically curated 16S rRNA database for cyanobacteria and enables comparisons against NCBI databases to facilitate the identification of rare prokaryotic strains in environmental samples. RASPAM is Findable, Accessible, Interoperable, and Reproducible (FAIR) and represents a robust tool for long-term monitoring of microbial communities in Antarctic and other extreme environments.