Profiling bacterial communities by MinION sequencing of ribosomal operons

Abstract Background An 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. Results To 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. Conclusions This 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

Arctic tundra soil bacterial communities active at subzero temperatures detected by stable isotope probing

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Identification of Bacteria Synthesizing Ribosomal RNA in Response to Uranium Addition During Biostimulation at the Rifle, CO Integrated Field Research Site

<div><p>Understanding which organisms are capable of reducing uranium at historically contaminated sites provides crucial information needed to evaluate treatment options and outcomes. One approach is determination of the bacteria which directly respond to uranium addition. In this study, uranium amendments were made to groundwater samples from a site of ongoing biostimulation with acetate. The active microbes in the planktonic phase were deduced by monitoring ribosomes production via RT-PCR. The results indicated several microorganisms were synthesizing ribosomes in proportion with uranium amendment up to 2 μM. Concentrations of U (VI) >2 μM were generally found to inhibit ribosome synthesis. Two active bacteria responding to uranium addition in the field were close relatives of <i>Desulfobacter postgateii</i> and <i>Geobacter bemidjiensis</i>. Since RNA content often increases with growth rate, our findings suggest it is possible to rapidly elucidate active bacteria responding to the addition of uranium in field samples and provides a more targeted approach to stimulate specific populations to enhance radionuclide reduction in contaminated sites.</p></div

Ribosomal response to uranium additions in groundwater after 24h for 4 OTUs (TRF-212, 213, 214, 215 bp) from wells D02 (open square), D07 (closed square), sampled in 2008, D01 (open circle), D08 (closed circle), sampled in 2009 (note the difference in scales).

<p>Ribosomal response to uranium additions in groundwater after 24h for 4 OTUs (TRF-212, 213, 214, 215 bp) from wells D02 (open square), D07 (closed square), sampled in 2008, D01 (open circle), D08 (closed circle), sampled in 2009 (note the difference in scales).</p

Comparison of RNA-TRFLP peak areas in frozen versus preserved river water field samples.

<p>Results are shown for riverine samples that were frozen, plotted against those preserved with BioDry and stored for either 10 or 30 d for the bacterial (A & B) & eukaryotic (C & D) communities. Values represent averages (n = 3 ± SD) of only those peaks present in both controls and BioDried samples; the number of OTUs used for each analysis is indicated at the bottom-right of each graph.</p

Percent total peak area from community DNA of 16S rRNA genes TRFLP profiles of pre-filters 2008 well D04 during field acetate injection: TRF 212-white, 213-light grey, 215-black, other <i>Geobacter</i>-like TRF’s-dark grey stipple, all other TRF peaks-light checked.

<p>Percent total peak area from community DNA of 16S rRNA genes TRFLP profiles of pre-filters 2008 well D04 during field acetate injection: TRF 212-white, 213-light grey, 215-black, other <i>Geobacter</i>-like TRF’s-dark grey stipple, all other TRF peaks-light checked.</p

Assessment of protein and <i>m</i>RNA integrity for seawater field samples preserved with BioDry.

<p>Gene expression for rbcL was assayed using clade-specific primers for diatoms (A) and haptophytes (B). Protein was assayed by western blot using an RbcL antibody (C). Red arrows represent assay positives for <i>Emiliania huxleyi</i> CCMP & Glycine max (soy) leaf extract respectively. Frozen controls (T₀), preserved samples (T₁₀ & T₃₀ days), as well as the representative DNA/contamination controls are indicated.</p

Comparison of RNA-TRFLP peak areas in frozen versus preserved seawater field samples.

<p>Results are shown for seawater samples that were frozen, plotted against those preserved with BioDry and stored for either 10 or 30 d for the bacterial (A & B), eukaryotic (C & D), or archaeal (E & F) communities. Values represent averages (n = 3 ± SD) of only those peaks present in both controls and BioDried samples; the number of OTUs used for each analysis is indicated at the bottom-right of each graph.</p

Chemical analysis of groundwater from well D04 during the 2008 field experiment-acetate (closed triangles) and bromide (open squares).

<p>The period of groundwater flush (shaded) and the times of biomass sampling (dotted line) are indicated. The chemical methods used in this analysis exhibit analytical variability in the femto-, nano-, and micro-molar range for uranium, iron/sulfide, and acetate respectively. All field measurements are beyond this range.</p