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.

瘤胃内活生物量占比30%-50%的各类原生动物，其生态作用至关重要但在很大程度上尚未被探明。由于单一物种培养难度极高，传统上显微镜观察是表征瘤胃原生动物最常用的手段，但该方法无法揭示它们在复杂瘤胃微生物组中的具体功能。过往为阐明其功能所开展的研究，主要聚焦于去除或富集原生动物群落所产生的宏观效应。近期研究已开始揭示原生动物与产甲烷古菌之间的关联，但受限于高通量分子研究方法的匮乏。相较于近期瘤胃原核生物与真菌的长读长DNA测序及宏基因组组装技术所取得的进展，瘤胃原生动物的同类基因组资源仍十分匮乏，这种差异或许暗示了在核酸提取、测序及后续分析环节存在研究偏向。本研究针对该问题，开发了一种从瘤胃液中富集瘤胃纤毛原生动物的方法，并进一步优化了DNA提取流程，可获得适用于长读长测序技术的高质量、高分子量DNA。本研究的富集流程与高分子量DNA提取方法相结合，有望大幅提升已测序原生动物基因组的数量，从而推动学界对这类神秘微生物的认知。