MiDAS 4.0: A comprehensive 16S rRNA reference database of bacteria in wastewater treatment systems across the globe

We recently developed a method that can produce millions of high-quality, full-length 16S rRNA gene sequences. This method was applied to samples collected at 667 wastewater treatment plants from 33 countries to create the first comprehensive, global 16S rRNA gene reference database for microbes in wastewater treatment systems (MiDAS 4). Sequences in MiDAS 4 were classified based on a new automated taxonomy classification algorithm (AutoTax), which provides a robust classification for all taxonomic ranks from kingdom to species-level for all sequences. AutoTax identified 8075 genera and 27204 species, revealing a much less diverse global community compared to what has previously been predicted. It also revealed some global distribution that partly depended on geography but also type of process design of the treatment plants. MiDAS4 also dramatically improves the specificity and reproducibility of taxonomic classification of amplicons, providing reliable community studies at sub-genus to sub-species-level. It also provides an opportunity to develop highly specific FISH probes that can be used to illuminate the morphological and functional diversity at the sub-genus level in situ. Since MiDAS 4 taxonomy provides names or placeholder names for nearly all microbes occurring in wastewater treatment systems across the globe, we anticipate that this will facilitate the sharing of knowledge across the scientific field.