This study explored the short-term planktonic microbial community structure and resilience in Lake Lanier (GA, USA) while simultaneously evaluating the technical aspects of identifying taxa via 16S rRNA gene amplicon and metagenomic sequence data. 16S rRNA gene amplicons generated from four temporally discrete samples were sequenced with 454 GS-FLX-Ti yielding ∼40,000 rRNA gene sequences from each sample and representing ∼300 observed OTUs. Replicates obtained from the same biological sample clustered together but several biases were observed, linked to either the PCR or sequencing-preparation steps. In comparisons with companion whole-community shotgun metagenome datasets, the estimated number of OTUs at each timepoint was concordant, but 1.5 times and ∼10 times as many phyla and genera, respectively, were identified in the metagenomes. Our analyses showed that the 16S rRNA gene captures broad shifts in community diversity over time, but with limited resolution and lower sensitivity compared to metagenomic data. We also identified OTUs that showed marked shifts in abundance over four close timepoints separated by perturbations and tracked these taxa in the metagenome vs. 16S rRNA amplicon data. A strong summer storm had less of an effect on community composition than did seasonal mixing, which revealed a distinct succession of organisms. This study provides insights into freshwater microbial communities and advances the approaches for assessing community diversity and dynamics in situ.

Next-generation sequencing (NGS) is commonly used in metagenomic studies of complex microbial communities but whether or not different NGS platforms recover the same diversity from a sample and their assembled sequences are of comparable quality remain unclear. We compared the two most frequently used platforms, the Roche 454 FLX Titanium and the Illumina Genome Analyzer (GA) II, on the same DNA sample obtained from a complex freshwater planktonic community. Despite the substantial differences in read length and sequencing protocols, the platforms provided a comparable view of the community sampled. For instance, derived assemblies overlapped in ∼90% of their total sequences and in situ abundances of genes and genotypes (estimated based on sequence coverage) correlated highly between the two platforms (R2>0.9). Evaluation of base-call error, frameshift frequency, and contig length suggested that Illumina offered equivalent, if not better, assemblies than Roche 454. The results from metagenomic samples were further validated against DNA samples of eighteen isolate genomes, which showed a range of genome sizes and G+C% content. We also provide quantitative estimates of the errors in gene and contig sequences assembled from datasets characterized by different levels of complexity and G+C% content. For instance, we noted that homopolymer-associated, single-base errors affected ∼1% of the protein sequences recovered in Illumina contigs of 10× coverage and 50% G+C; this frequency increased to ∼3% when non-homopolymer errors were also considered. Collectively, our results should serve as a useful practical guide for choosing proper sampling strategies and data possessing protocols for future metagenomic studies.

SummaryBacteria of the SAR11 clade constitute up to one half of all microbial cells in the oxygen-rich surface ocean. DNA sequences from SAR11 are also abundant in oxygen minimum zones (OMZs) where oxygen falls below detection and anaerobic microbes play important roles in converting bioavailable nitrogen to N2 gas. Evidence for anaerobic metabolism in SAR11 has not yet been observed, and the question of how these bacteria contribute to OMZ biogeochemical cycling is unanswered. Here, we identify the metabolic basis for SAR11 activity in anoxic ocean waters. Genomic analysis of single cells from the world’s largest OMZ revealed diverse and previously uncharacterized SAR11 lineages that peak in abundance at anoxic depths, but are largely undetectable in oxygen-rich ocean regions. OMZ SAR11 contain adaptations to low oxygen, including genes for respiratory nitrate reductases (Nar). SAR11 nar genes were experimentally verified to encode proteins catalyzing the nitrite-producing first step of denitrification and constituted ~40% of all OMZ nar transcripts, with transcription peaking in the zone of maximum nitrate reduction rates. These results redefine the ecological niche of Earth’s most abundant organismal group and suggest an important contribution of SAR11 to nitrite production in OMZs, and thus to pathways of ocean nitrogen loss.

Lake Lanier is an important freshwater lake for the southeast United States, as it represents the main source of drinking water for the Atlanta metropolitan area and is popular for recreational activities. Temperate freshwater lakes such as Lake Lanier are underrepresented among the growing number of environmental metagenomic data sets, and little is known about how functional gene content in freshwater communities relates to that of other ecosystems. To better characterize the gene content and variability of this freshwater planktonic microbial community, we sequenced several samples obtained around a strong summer storm event and during the fall water mixing using a random whole-genome shotgun (WGS) approach. Comparative metagenomics revealed that the gene content was relatively stable over time and more related to that of another freshwater lake and the surface ocean than to soil. However, the phylogenetic diversity of Lake Lanier communities was distinct from that of soil and marine communities. We identified several important genomic adaptations that account for these findings, such as the use of potassium (as opposed to sodium) osmoregulators by freshwater organisms and differences in the community average genome size. We show that the lake community is predominantly composed of sequence-discrete populations and describe a simple method to assess community complexity based on population richness and evenness and to determine the sequencing effort required to cover diversity in a sample. This study provides the first comprehensive analysis of the genetic diversity and metabolic potential of a temperate planktonic freshwater community and advances approaches for comparative metagenomics.

Assembling individual genomes from complex community metagenomic data remains a challenging issue for environmental studies. We evaluated the quality of genome assemblies from community short read data (Illumina 100 bp pair-ended sequences) using datasets recovered from freshwater and soil microbial communities as well as in silico simulations. Our analyses revealed that the genome of a single genotype (or species) can be accurately assembled from a complex metagenome when it shows at least about 20 Â coverage. At lower coverage, however, the derived assemblies contained a substantial fraction of non-target sequences (chimeras), which explains, at least in part, the higher number of hypothetical genes recovered in metagenomic relative to genomic projects. We also provide examples of how to detect intrapopulation structure in metagenomic datasets and estimate the type and frequency of errors in assembled genes and contigs from datasets of varied species complexity.