Metagenomics-Toolkit: the flexible and efficient cloud-based metagenomics workflow featuring machine learning-enabled...

The metagenome analysis of complex environments with thousands of datasets, such as those in the Sequence Read Archive, requires substantial computational resources for it to be completed within a reasonable time frame. Efficient use of infrastructure is essential, and analyses must be fully reproducible with publicly available workflows to ensure transparency. Here, we introduce the Metagenomics-Toolkit, a scalable, data-agnostic workflow that automates the analysis of short and long metagenomic reads from Illumina and Oxford Nanopore Technology devices, respectively. The Metagenomics-Toolkit provides standard features such as quality control, assembly, binning, and annotation, along with unique capabilities including plasmid identification, recovery of unassembled microbial community members, and discovery of microbial interdependencies through dereplication, co-occurrence, and genome-scale metabolic modeling. Additionally, the Metagenomics-Toolkit includes a machine learning-optimized assembly step that adjusts peak RAM usage to match actual requirements, reducing the need for high-memory hardware. It can be executed on user workstations and includes optimizations for efficient cloud-based cluster execution. We compare the Metagenomics-Toolkit with five widely used metagenomics workflows and demonstrate its capabilities on 757 sewage metagenome datasets to investigate a possible sewage core microbiome. The Metagenomics-Toolkit is open source and available at https://github.com/metagenomics/metagenomics-tk.