Computational protocol: Abundance and Diversity of Denitrifying and Anammox Bacteria in Seasonally Hypoxic and Sulfidic Sediments of the Saline Lake Grevelingen

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Protocol publication

[…] Sequences were analyzed for the presence of chimeras using the Bellerophon tool at the GreenGenes website ( Sequences were aligned with MEGA6 software (Tamura et al., ) by using the alignment method ClustalW. The phylogenetic trees of the nirS and aprA genes were computed with the Neighbor-Joining method (Saitou and Nei, ) using the Poisson model with a bootstrap test of 1000 replicates. The phylogenetic affiliation of the partial anammox bacteria 16S rRNA gene sequences was compared to release 123 of the SILVA NR SSU Ref database (; Quast et al., ) using the ARB software package (Ludwig et al., ). Sequences were added to the reference tree supplied by the SILVA database using the ARB Parsimony tool. Sequences were deposited in NCBI with the following accession numbers: KP886533–KP886678 for nirS gene sequences of denitrifiers, KP886679–KP8866700 for 16S rRNA gene sequences of anammox bacteria, KP886701–KP886721 for nirS gene sequences of anammox bacteria and KP886722–KP886804 for aprA gene sequences of SOB and SRB. [...] qPCR analyses were performed on a Biorad CFX96™ Real-Time System/C1000 Thermal cycler equipped with the CFX Manager™ software for sediment DNA/RNA extracts (S1, S2, and S3; 0–5 cm sediment depth; 1 cm resolution). Detailed information about the primers used in this study are summarized in Table . The abundance of denitrifying bacteria specific nirS gene was quantified using the primer set nirS1F/nirS3R as described by Braker and Fesefeldt (). The abundance of anammox bacteria 16S rRNA gene was estimated using primers Brod541F/Amx820R as described by Li et al. (). Additionally, a fragment of the Scalindua sp. specific nirS gene, which codes for the cytochrome cd1-containing nitrite reductase, was quantified using the primer combination Scnir372F/Scnir845R as described by Lam et al. (). The abundance of SOB and SRB including sulfide dependent denitrifiers was estimated by targeting the dissimilatory adenosine-5′-phosphosulfate (APS) reductase (aprA gene) involved in the APS reduction of SOB and in the sulfite oxidation of sulfate-reducing bacteria by using the primer combination Apr-1-FW/Apr-5-RW as described by Meyer and Kuever () (see Table for details). Gene abundances are expressed as copies g−1 sediment of wet weight.All qPCR amplifications were performed in triplicate with standard curves ranging from 100 to 107 molecules per microliter. Standard curves and qPCR amplifications were performed as previously described by Lipsewers et al. (). Coefficients of determination (R2) for standard curves ≥0.998 and qPCR efficiencies (E) ≥80% were accepted. […]

Pipeline specifications

Software tools MEGA, Clustal W, ARB, CFX Manager, BRAKER
Applications Phylogenetics, Transcription analysis
Chemicals Carbon, Nitrogen, Oxygen, Sulfur