Method evaluation for viruses in activated sludge: Concentration, sequencing, and identification.

Activated sludge (AS) in wastewater treatment plants is one of the largest artificial microbial ecosystems on earth and it makes enormous contributions to human societies. Viruses are an important component in AS with a high abundance. However, their communities and functionalities have not been as widely explored as those of other microorganisms, such as bacteria. This gap is mainly due to technical challenges in effective viral concentration, extraction, and sequencing. In this study, we compared four kinds of concentration methods, two sequencing approaches, and four identification bioinformatic tools to evaluate the whole analysis workflow for viruses in AS. Results showed flocculation, filtration, and resuspension (FFR) could get the longest DNA lengths and ultracentrifugation obtained the highest DNA yields for viruses in AS. Based on the results of present study, FFR and tangential flow filtration with the membrane pore size of 100 kDa were most recommended to concentrate viruses in AS samples with huge volumes. Besides, different concentration methods could get different viral catalogs and thus multiple methods should be combined to get the whole picture of viruses in the system. In addition, geNomad was the most recommended identification tool for viruses in the present study and the long-read sequencing could improve the assembly statistics of viruses when compared with the short-read sequencing. For the 8192 viral operational taxonomic units in this study, 95.1 % of them were phages and belonged to the same lineage at the order level of Caudovirales. Virulent phages dominated the AS system and Pseudomonadota were the main host. Taken together, this study provides new insights into methods selection for virus research of AS.