New long read sequencing technologies offer huge potential for effective recovery of complete, closed genomes from complex microbial communities. Using long read data (ONT MinION) obtained from an ensemble of activated sludge enrichment bioreactors we recover 22 closed or complete genomes of community members, including several species known to play key functional roles in wastewater bioprocesses, specifically microbes known to exhibit the polyphosphate- and glycogen-accumulating organism phenotypes (namely Candidatus Accumulibacter and Dechloromonas, and Micropruina, Defluviicoccus and Candidatus Contendobacter, respectively), and filamentous bacteria (Thiothrix) associated with the formation and stability of activated sludge flocs. Additionally we demonstrate the recovery of close to 100 circularised plasmids, phages and small microbial genomes from these microbial communities using long read assembled sequence. We describe methods for validating long read assembled genomes using their counterpart short read metagenome-assembled genomes, and assess the influence of different correction procedures on genome quality and predicted gene quality. Our findings establish the feasibility of performing long read metagenome-assembled genome recovery for both chromosomal and non-chromosomal replicons, and demonstrate the value of parallel sampling of moderately complex enrichment communities to obtaining high quality reference genomes of key functional species relevant for wastewater bioprocesses.

新的长读长测序技术为从复杂的微生物群落中有效恢复完整、封闭的基因组提供了巨大的潜力。使用从一组活性污泥富集生物反应器中获得的长读数据 (ONT MinION)，我们恢复了 22 个社区成员的封闭或完整基因组，包括已知在废水生物过程中发挥关键功能作用的几种物种，特别是已知表现出聚磷酸盐和糖原积累生物体表型（分别为Candidatus Accumulibacter 和Dechloromonas以及Micropruina、Defluviicoccus和Candidatus Contendobacter）和丝状细菌（Thiothrix) 与活性污泥絮凝体的形成和稳定性有关。此外，我们还展示了使用长读长组装序列从这些微生物群落中回收近 100 个环化质粒、噬菌体和小型微生物基因组。我们描述了使用对应的短读宏基因组组装基因组验证长读组装基因组的方法，并评估了不同校正程序对基因组质量和预测基因质量的影响。我们的研究结果确立了对染色体和非染色体复制子执行长读长宏基因组组装基因组恢复的可行性，并证明了中等复杂富集群落平行采样对于获得与废水生物过程相关的关键功能物种的高质量参考基因组的价值。