Browsing UA Faculty Research by Subjects
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16S rRNA gene sequencing on a benchtop sequencer: accuracy for identification of clinically important bacteria.Test the choice of 16S rRNA gene amplicon and data analysis method on the accuracy of identification of clinically important bacteria utilizing a benchtop sequencer. Nine 16S rRNA amplicons were tested on an Ion Torrent PGM to identify 41 strains of clinical importance. The V1-V2 region identified 40 of 41 isolates to the species level. Three data analysis methods were tested, finding that the Ribosomal Database Project's SequenceMatch outperformed BLAST and the Ion Reporter Metagenomics analysis pipeline. Lastly, 16S rRNA gene sequencing mixtures of four species through a six log range of dilution showed species were identifiable even when present as 0·1% of the mixture. Sequencing the V1-V2 16S rRNA gene region, made possible by the increased read length Ion Torrent PGM sequencer's 400 base pair chemistry, may be a better choice over other commonly used regions for identifying clinically important bacteria. In addition, the SequenceMatch algorithm, freely available from the Ribosomal Database Project, is a good choice for matching filtered reads to organisms. Lastly, 16S rRNA gene sequencing's sensitivity to the presence of a bacterial species at 0·1% of a mixture suggests it has sufficient sensitivity for samples in which important bacteria may be rare. We have validated 16S rRNA gene sequencing on a benchtop sequencer including simple mixtures of organisms; however, our results highlight deficits for clinical application in place of current identification methods.
Influence of periparturient and postpartum diets on rumen methanogen communities in three breeds of primiparous dairy cowsBackground: Enteric methane from rumen methanogens is responsible for 25.9 % of total methane emissions in the United States. Rumen methanogens also contribute to decreased animal feed efficiency. For methane mitigation strategies to be successful, it is important to establish which factors influence the rumen methanogen community and rumen volatile fatty acids (VFA). In the present study, we used next-generation sequencing to determine if dairy breed and/or days in milk (DIM) (high-fiber periparturient versus high-starch postpartum diets) affect the rumen environment and methanogen community of primiparous Holstein, Jersey, and Holstein-Jersey crossbreeds. Results: When the 16S rRNA gene sequences were processed and assigned to operational taxonomic units (OTU), a core methanogen community was identified, consisting of Methanobrevibacter (Mbr.) smithii, Mbr. thaueri, Mbr. ruminantium, and Mbr. millerae. The 16S rRNA gene sequence reads clustered at 3 DIM, but not by breed. At 3 DIM, the mean % abundance of Mbr. thaueri was lower in Jerseys (26.9 %) and higher in Holsteins (30.7 %) and Holstein-Jersey crossbreeds (30.3 %) (P < 0.001). The molar concentrations of total VFA were higher at 3 DIM than at 93, 183, and 273 DIM, whereas the molar proportions of propionate were increased at 3 and 93 DIM, relative to 183 and 273 DIM. Rumen methanogen densities, distributions of the Mbr. species, and VFA molar proportions did not differ by breed. Conclusions: The data from the present study suggest that a core methanogen community is present among dairy breeds, through out a lactation. Furthermore, the methanogen communities were more influenced by DIM and the breed by DIM interactions than breed differences.
Organic amendments change soil organic C structure and microbial community but not total organic matter on sub-decadal scalesOrganic C has many benefits for soil, but it is depleted by tillage and crop harvest, and especially so for biofuel crops. Accordingly, strategies such as partially retaining stover or planting a cover crop can help ameliorate the negative effect of C removal. We used a long-term field experiment to study the impacts of stover retention and planting a cover crop on soil organic matter (SOM), its extractable components, and the soil microbial community. SOM chemical composition characterization was determined by electrospray ionization (ESI) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) in sequential water, methanol (MeOH), and chloroform (CHCl3) extracts. The characteristics of the soil bacterial community were measured by phospholipid fatty acid (PLFA), real-time quantitative PCR, and 16S rRNA gene sequence. The variations in total SOM content, total microbial biomass, and bacterial population were slight among treatments, but SOM chemical compounds, arbuscular mycorrhizal fungi (AMF) biomass, and bacterial structure changed significantly, and especially so in the coupled application of stover retention and cover crop. Specifically, stover retention enriched more lignin-like compounds in soil, whereas cover crop enriched more condensed hydrocarbons, and had more compounds with an aromaticity index (AI) > 0.5. The bacterial community was not altered by the cover crop, but the corn stover retention increased the relative abundances of Myxococcales (Deltaproteobacteria) and decreased that of Actinobacteria. Redundancy analysis (RDA) further revealed that the bacterial community in the stover treatments had a significant positive association with CHCl3-extracted chemical classes, i.e. unsaturated hydrocarbons and lipids, with the coupled application (stover and cover crop), and lignin and proteins with the corn stover only treatment. Taken together, our study shows how different C addition practices influence the molecular composition of SOM and the structure of soil microbial communities.