Effects of enrichment and DNA extraction method on long-read sequencing of rumen ciliate protozoa

Different species of protozoa, constituting 30-50% of the living biomass in the rumen, have a significant but largely unexplored role. Traditionally, microscopy has been the most useful method for characterizing rumen protozoa, given the challenges of culturing individual species. However, this method cannot reveal their functions within the complex rumen microbiome. Past efforts to elucidate their functional role have primarily focused on the broad effects of removing or enriching the protozoan community. Recent studies have started to uncover the link between protozoa and methane-producing archaea, but they are hindered by a lack of high-throughput molecular methods. In contrast to recent advances in long-read DNA sequencing and metagenome assembly for rumen prokaryotes and fungi, similar genomic resources for rumen protozoa are lacking. This discrepancy may suggest a bias in extraction, sequencing, or downstream analysis. Here, we address this by introducing a method to enrich rumen ciliate protozoa from rumen fluid and further develop a DNA extraction method to produce high-quality, high-molecular-weight DNA suitable for long-read sequencing technologies. The combination of our enrichment process and methodology for extracting high-molecular-weight DNA has the potential to significantly increase the number of sequenced protozoal genomes, thereby advancing our understanding of these enigmatic microbes.