BackgroundAn approach utilizing the long-read capability of the Oxford Nanopore MinION to rapidly sequence bacterial ribosomal operons of complex natural communities was developed. Microbial fingerprinting employs domain-specific forward primers (16S rRNA subunit), reverse primers (23S rRNA subunit), and a high-fidelity Taq polymerase with proofreading capabilities. Amplicons contained both ribosomal subunits for broad-based phylogenetic assignment (~ 3900 bp of sequence), plus the intergenic spacer (ITS) region (~ 300 bp) for potential strain-specific identification.ResultsTo test the approach, bacterial rRNA operons (~ 4200 bp) were amplified from six DNA samples employing a mixture of farm soil and bioreactor DNA in known concentrations. Each DNA sample mixture was barcoded, sequenced in quadruplicate (n = 24), on two separate 6-h runs using the MinION system (R7.3 flow cell; MAP005 and 006 chemistry). From nearly 90,000 MinION reads, roughly 33,000 forward and reverse sequences were obtained. This yielded over 10,000 2D sequences which were analyzed using a simplified data analysis pipeline based on NCBI Blast and assembly with Geneious software. The method could detect over 1000 operational taxonomic units in the sample sets in a quantitative manner. Global sequence coverage for the various rRNA operons ranged from 1 to 1951x. An iterative assembly scheme was developed to reconstruct those rRNA operons with > 35x coverage from a set of 30 operational taxonomic units (OTUs) among the Proteobacteria, Actinobacteria, Acidobacteria, Firmicutes, and Gemmatimonadetes. Phylogenetic analysis of the 16S rRNA and 23S rRNA genes from each operon demonstrated similar tree topologies with species/strain-level resolution.ConclusionsThis sequencing method represents a cost-effective way to profile microbial communities. Because the MinION is small, portable, and runs on a laptop, the possibility of microbiota characterization in the field or on robotic platforms becomes realistic.Electronic supplementary materialThe online version of this article (10.1186/s40168-017-0336-9) contains supplementary material, which is available to authorized users.

The atmosphere contains diverse living microbes, of which the heterotrophic community has been the most studied. Microbes with other trophic modes, such as photoautotrophy, have received much less attention. Here, culture-independent and dependent methods were used to examine the presence and diversity of oxygenic photoautotrophic microbes in clouds and rain collected at or around puy de Dôme Mountain, central France. Cloud water was collected from the summit of puy de Dôme (1465 m above sea level (a.s.l.)) for cultivation and metagenomic analysis. Cyanobacteria, diatoms, green algae, and other oxygenic photoautotrophs were found to be recurrent members of clouds, while green algae affiliated with the Chlorellaceae were successfully cultured from three different clouds. Additionally, rain samples were collected below the mountain from Opme meteorological station (680 m, a.s.l.). The abundance of chlorophyll-a containing cells and the diversity of cyanobacteria and green algae in rain were assessed by flow cytometry and amplicon sequencing. The corresponding downward flux of chlorophyll-a containing organisms to the ground, entering surface ecosystems with rain, varied with time and was estimated between ∼1 and >300 cells cm-2 day-1 during the sampling period. Besides abundant pollen from Pinales and Rosales, cyanobacteria of the Chroococcidiopsidales and green algae of the Trebouxiales were dominant in rain samples. Certain members of these taxa are known to be ubiquitous and stress-tolerant and could use the atmosphere for dispersal. Overall, our results indicate that the atmosphere carries diverse, viable oxygenic photoautotrophic microbes and acts as a dispersal vector for this microbial guild. IMPORTANCE: Information regarding the diversity and abundance of oxygenic photoautotrophs in the atmosphere is limited. More information from diverse locations is needed. These airborne organisms could have important impacts upon atmospheric processes and on the ecosystems they enter after deposition. Oxygenic photoautotrophic microbes are integral to ecosystem functioning and some have the potential to affect human health. A better understanding of the diversity and the movements of these aeolian dispersed organisms is needed to understand their ecology, as well as how they could affect ecosystems and human health.

Bioaerosols have impacts on atmospheric processes, as well as ecosystem and human health. Common bioaerosol collection methods include impaction, liquid impingement, filtration, and electrostatic precipitation. These methods are used by active samplers that require an air mover and power, but this requirement can also represent a major constraint in field studies. Alternatively, passive samplers do not require power and can operate for long times. In this study, the Rutgers Electrostatic Passive Sampler (REPS), which captures particles by electrostatic attraction and gravitational settling, was deployed at the summit of puy de Dôme (1465 m a.s.l., France) alongside an active PM10 sampler (~1000 L min -1 ) collecting aerosols on a quartz fiber filter. The diversity of the airborne bacteria captured by both samplers across six weekly sampling periods was examined by 16S rRNA gene amplicon sequencing. The dominant phyla observed by both samplers were similar and included Firmicutes, Proteobacteria, and Actinobacteriota. Overall, 12 to 63% of the total bacterial richness at the genus level was shared between the two samplers, depending upon a paired sample, i.e., sampling week. The PM10 sampler and REPS detected the same dominant genera, including Lysinibacillus and Sphingomonas, although their relative abundances for each paired sampler varied. The observed bacterial richness and diversity, as estimated through Shannon's and Simpson's indexes, were significantly greater in REPS samples compared to the PM10 samples. The results suggest that REPS could be used for simple and convenient sampling of bioaerosols, especially in remote areas and other locations with limited power access.

Currently, the cellular activities of bacteria in the airborne state outside of water droplets have not been heavily studied. Evidence suggests that these airborne bacteria produce ribosomes and metabolize gaseous compounds.