Computational protocol: Characterisation of the effect of a simulated hydrocarbon spill on diazotrophs in mangrove sediment mesocosm

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[…] Four samples were selected for nifH based clone library analysis. These were: Polluted + oil, T-0 (Poll T-0), Polluted + oil, T-75 (Poll T-75), Pristine + oil, T-0 (Prist T-0) and Pristine + oil, T-75 (Prist T-75). The nifH genes from total community DNA were amplified using primers FGPH19 (Simonet et al. ) and PolR (Poly et al. ) as previously described (Diallo et al. ). The PCR reaction mix (25 μL) contained 10.2 mM Tris, 2.5 mM MgCl2, 50 mM KCl, 0.2 mM of each dNTP, 0.5 μM of each primer and 2.5 U of Taq DNA polymerase. PCR was carried out on a Perkin–Elmer GeneAmp PCR System 9700 (Perkin–Elmer Applied Biosystems, Nieuwerkerk a/d IJsel, the Netherlands) and thermal cycling was as follows: 95°C for 5 min; 30 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 1 min with a final elongation step of 72°C for 7 min. These 320 bp PCR products were ligated into the pGEM-T easy vector (Promega, Madison, USA) according to the manufacturer’s protocol. The ligation products were introduced into Escherichia coli MM294 competent cells (Sylphium Life Sciences, Groningen, The Netherlands) by transformation according to the supplier’s protocol, after which cells were plated onto selective media (Sambrook and Russell, ). Following growth at 37°C, white colonies were randomly picked, and plasmids with inserts of the correct size were isolated using the Cetyl Trimethyl Ammonium Bromide (CTAB) isolation method (Sambrook and Russell ). Sequencing reactions were performed on the plasmid material according to the Perkin–Elmer ABI Prism protocol (Applied Biosystems, Foster City, USA) using primer SP6. Sequence runs were done on an ABI377 DNA sequencer (Applied Biosystems, Foster City, USA).All chromatograms were analysed for sequence quality using Bioedit (Hall ). Sequences containing ambiguities were not further analysed. Sequences were searched for vector contamination in NCBI’s VecScreen (http://www.ncbi.nlm.nih.gov/VecScreen/VecScreen.html) and contaminant sequences were omitted. The sequences were then checked for the presence of chimeras using the Greengenes chimera-check tool (http://greengenes.lbl.gov/cgi-bin/nph-bel3_interface.cgi, [DeSantis et al. ]). The non-chimeric non-vector sequences were then classified based on the most similar sequence from a cultivated organism using the BLAST-n tool from NCBI (Altschul et al. ). When no cultivated organism was found between the 50 best matches the best match was then used for classification. The sequences obtained were aligned using ClustalX (Thompson et al. ) and the output file was used in Phylip 3.66 to construct a distance matrix using the Jukes and Cantor distance model (Jukes and Cantor, ). These were then used in the software DOTUR (Schloss and Handelsman ) to identify operational taxonomic units (OTUs) and generate estimations of richness and diversity, as well as rarefaction curves. A Neighbor-Joining tree of 86 nifH sequences was constructed using MEGA 4.0. The bootstrap consensus tree inferred from 1,000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The sequences reported in this study were deposited on GenBank under the accession numbers FJ669424-FJ669486. […]

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