Project description:High-throughput metabarcoding studies on fungi and other eukaryotic microorganisms are rapidly becoming more frequent and more complex, requiring researchers to handle ever increasing amounts of raw sequence data. Here, we provide a flexible pipeline for pruning and analyzing fungal barcode (ITS rDNA) data generated as paired-end reads on Illumina MiSeq sequencers. The pipeline presented includes specific steps fine-tuned for ITS, that are mostly missing from pipelines developed for prokaryotes. It (1) employs state of the art programs and follows best practices in fungal high-throughput metabarcoding; (2) consists of modules and scripts easily modifiable by the user to ensure maximum flexibility with regard to specific needs of a project or future methodological developments; and (3) is straightforward to use, also in classroom settings. We provide detailed descriptions and revision techniques for each step, thus giving the user maximum control over data treatment and avoiding a black-box approach. Employing this pipeline will improve and speed up the tedious and error-prone process of cleaning fungal Illumina metabarcoding data.
Project description:Peatlands of the Lehstenbach catchment (Germany) house so far unidentified microorganisms with phylogenetically novel variants of the dissimilatory (bi)sulfite reductase genes dsrAB. These genes are characteristic for microorganisms that reduce sulfate, sulfite, or some organosulfonates for energy conservation, but can also be present in anaerobic syntrophs. However, nothing is currently known regarding the abundance, community dynamics, and biogeography of these dsrAB-carrying microorganisms in peatlands. To tackle these issues, soils from a Lehstenbach catchment site (Schlöppnerbrunnen II fen) from different depths were sampled at three time points over a six-year period to analyze the diversity and distribution of dsrAB-containing microorganisms by a newly developed functional gene microarray and quantitative PCR assays. Members of novel, uncultivated dsrAB lineages (approximately representing species-level groups) (i) dominated a temporally stable but spatially structured dsrAB community and (ii) represented ‘core’ members (up to 1-1.7% relative abundance) of the autochthonous microbial community in this fen. In addition, denaturing gradient gel electrophoresis (DGGE)- and clone library-based comparison of the dsrAB diversity in soils from a wet meadow, three bogs, and five fens of various geographic locations (distance ~1-400 km), identified one Syntrophobacter-related and nine novel dsrAB lineages to be widespread in low-sulfate peatlands. Signatures of biogeography in dsrB-DGGE data were not correlated with geographic distance but could largely be explained by soil pH and wetland type, implying that distribution of dsrAB-carrying microorganisms in wetlands on the scale of a few hundred kilometers is not limited by dispersal but determined by contemporary environmental conditions. 36 dsrAB clones for chip evaluation, 33 hybridizations of labeled dsrAB RNA from environmental peatsoil samples
Project description:Peatlands of the Lehstenbach catchment (Germany) house so far unidentified microorganisms with phylogenetically novel variants of the dissimilatory (bi)sulfite reductase genes dsrAB. These genes are characteristic for microorganisms that reduce sulfate, sulfite, or some organosulfonates for energy conservation, but can also be present in anaerobic syntrophs. However, nothing is currently known regarding the abundance, community dynamics, and biogeography of these dsrAB-carrying microorganisms in peatlands. To tackle these issues, soils from a Lehstenbach catchment site (Schlöppnerbrunnen II fen) from different depths were sampled at three time points over a six-year period to analyze the diversity and distribution of dsrAB-containing microorganisms by a newly developed functional gene microarray and quantitative PCR assays. Members of novel, uncultivated dsrAB lineages (approximately representing species-level groups) (i) dominated a temporally stable but spatially structured dsrAB community and (ii) represented ‘core’ members (up to 1-1.7% relative abundance) of the autochthonous microbial community in this fen. In addition, denaturing gradient gel electrophoresis (DGGE)- and clone library-based comparison of the dsrAB diversity in soils from a wet meadow, three bogs, and five fens of various geographic locations (distance ~1-400 km), identified one Syntrophobacter-related and nine novel dsrAB lineages to be widespread in low-sulfate peatlands. Signatures of biogeography in dsrB-DGGE data were not correlated with geographic distance but could largely be explained by soil pH and wetland type, implying that distribution of dsrAB-carrying microorganisms in wetlands on the scale of a few hundred kilometers is not limited by dispersal but determined by contemporary environmental conditions.