Do you cov me? Effect of coverage reduction on metagenome shotgun sequencing studies [version 4; peer review: 2 approved with reservations, 2 not approved]

Shotgun metagenomics sequencing is a powerful tool for the characterization of complex biological matrices, enabling analysis of prokaryotic and eukaryotic organisms and viruses in a single experiment, with the possibility of reconstructing de novo the whole metagenome or a set of genes of interest. One of the main factors limiting the use of shotgun metagenomics on wide scale projects is the high cost associated with the approach. We set out to determine if it is possible to use shallow shotgun metagenomics to characterize complex biological matrices while reducing costs. We used a staggered mock community to estimate the optimal threshold for species detection. We measured the variation of several summary statistics simulating a decrease in sequencing depth by randomly subsampling a number of reads. The main statistics that were compared are diversity estimates, species abundance, and ability of reconstructing de novo the metagenome in terms of length and completeness. Our results show that diversity indices of complex prokaryotic, eukaryotic and viral communities can be accurately estimated with 500,000 reads or less, although particularly complex samples may require 1,000,000 reads. On the contrary, any task involving the reconstruction of the metagenome performed poorly, even with the largest simulated subsample (1,000,000 reads). The length of the reconstructed assembly was smaller than the length obtained with the full dataset, and the proportion of conserved genes that were identified in the meta-genome was drastically reduced compared to the full sample. Shallow shotgun metagenomics can be a useful tool to describe the structure of complex matrices, but it is not adequate to reconstruct—even partially—the metagenome.