Background: Anaerobic digestion (AD) has long been critical technology for green energy, but the majority of the microorganisms involved are unknown and are currently not cultivable, which makes abundance tracking difficult. Developments in nanopore long-read sequencing make it a promising approach for monitoring microbial communities via metagenomic sequencing. For reliable monitoring of AD via long reads, a robust protocol for obtaining less fragmented, high-quality DNA, while preserving

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... ria and archaea composition, was established. Results: Samples from 20 different biogas/wastewater reactors were investigated, and a median of 20.5 Gb sequencing data per nanopore flow cell was retrieved for each reactor using the developed DNA isolation protocol. The nanopore sequencing data was compared against Illumina sequencing data while using different taxonomic indices for read classifications. The Genome Taxonomy Database (GTDB) index allowed sufficient characterisation of the abundance of bacteria and archaea in biogas reactors with a dramatic improvement (1.8- to 13-fold increase) in taxonomic classification compared to the RefSeq index. Both technologies performed similarly in taxonomic read classification with a slight advantage for Illumina in regards to the total proportion of classified reads. However, nanopore sequencing data revealed a higher genus richness after classification. Conclusion: Metagenomic read classification via nanopore provides a promising approach to monitor the abundance of taxa present in a microbial AD community, as an alternative to 16S rRNA studies or Illumina Sequencing.