Estimation of metagenome size and structure in an experimental soil microbiota from low coverage next-generation sequence data.

AIMS A major challenge in metagenome studies is to estimate the true size of all combined genomes. Here, we present a novel approach to estimate the size of all combined genomes for low coverage next-generation sequencing (NGS) data through empirically determined copy numbers of random DNA fragments. METHODS AND RESULTS Size estimates were made based on analyses of two experimental soil micro-ecosystems - simulating soil with and without earthworms. Our analyses showed combined genome sizes of about log 11 nucleotides for each of the soil micro-ecosystems, as estimated from qPCR determined copy numbers of random DNA fragments. This corresponds to more than 20000 unique bacterial genomes in each sample. There seemed, however, to be a bacterial subpopulation in the earthworm soil, not being present in the nonearthworm soil. To describe the structure of the metagenomes, both total DNA and amplified 16S rRNA gene sequence libraries were generated with 454-sequencing. Bioinformatic analysis of 454 sequence libraries showed a large functional but low taxonomic overlap between the samples with and without earthworms. A neutrality test indicated that rare species have a competitive advantage over abundant species in both micro-ecosystems providing a potential explanation for the large metagenome sizes. CONCLUSIONS We have shown that the soil metagenome is very large and that the large size is probably a consequence of top-down selection of the dominant bacterial species. SIGNIFICANCE AND IMPACT OF THE STUDY Estimates of metagenome size from low coverage NGS data will be important for guiding future NGS set-ups.