Benchmarking of shotgun sequencing depth highlights strain-level limitations of metagenomic analysis

Shallow metagenomics promises taxonomic and functional insights into samples at an affordable price. To determine the depth of sequencing required for specific analysis, benchmarking is required using defined communities. We used complex mixtures of DNA from cultured gut bacteria and analysed taxonomic composition, strain-level resolution, and functional profiles at nine sequencing depths (0.1-10.0 Gb). Reference-based analysis provided accurate taxonomic, and strain-level insights at 0.5-1.0 Gb. In contrast, de-novo metagenome-assembled genome (MAG) reconstruction required deep sequencing (>10 Gb). Furthermore, it was found that even high-quality MAGs were chimeric, with few (54.5 %) accurately representing the original strains. Functionally, 2 Gb provided reliable insights at the pathway level, but sufficient proteome coverage was only achieved at 10 Gb. Library preparation and host-DNA were identified as confounders in shallow metagenomic analysis. This comprehensive analysis using complex mock communities provides guidance to an increasing community of scientists interested in using shallow metagenomics, and highlights the limitations of MAGs in capturing strain-level diversity.