Time and space resolved deep metagenomics to investigate selection pressures on low abundant species in complex environments

Metagenomics offers great potential for investigation of species and strain diversification over time. Recent progress in sequencing technologies have made it possible to investigate species diversification and niche adaptation in low abundant populations (<1%) in complex environments by sequencing hundreds of gigabases of DNA sequence. However, lack of replication and time series data as well as DNA extraction independent validation have limited the generalization of most earlier studies. For a period of 6 years we have studied community dynamics and operational performance in 25 Danish Enhanced Biological Phosphorus Removal (EBPR) wastewater treatment plants using qFISH and 16S sequencing and identified a surprisingly stable core community. This makes EBPR a prime target for metagenomic investigations of species diversification over time and between plants. The aim of the project was to investigate time and space resolved diversity of key species in the core EBPR community using available reference genomes and genomes assembled directly from metagenomes.
A total of 500 Gb sequence was generated using Illumina HiSeq2000 for metagenomics and V4 16S rRNA gene sequencing. To track changes over time and between EBPR plants we sequenced a total of 10 samples from 3 different plants over a 3 year period at a depth of 25 Gb each. In addition, one time point was selected for deep sequencing, generating 200 Gb of sequence divided between replicates. Quantitative FISH analysis using >30 oligonucleotide probes was carried out. Data analysis was carried out largely as described in Albertsen et al., 2011 (ISME J doi:10.1038/ismej.2011.176).
The DNA extraction method was optimized based on 16S rRNA and qFISH results and showed the need for optimizing the extraction method depending on the microorganisms present. Individual assemblies of the replicate data sets did not reveal major changes in gene content, although differences were present The deep metagenome made it possible to assemble several draft genomes of functional important genus' including the actinobacterial Tetrasphaera and the nitrifier Nitrospira. By mapping the raw reads to reference genomes and de novo assembled draft genomes it was possible to track changes in gene content and mutation frequency over time and between plants. The metagenome analysis of micro-diversity revealed large differences in the degree of micro-diversity within different groups of bacteria, also over time. Many of these findings could be related to niche breadth. A common core gene pool could be identified in each bacterial group, but with large differences - mainly related to phage defense. It is interesting that the observed diversity could not be resolved using FISH or V4 16S rRNA sequencing where the populations seemed much more stable. This information can be used to expand the current ecosystem models used in EBPR and contribute to the understanding of ecosystem dynamics in complex environments.