Computational protocol: Culture-Dependent and -Independent Identification of Polyphosphate-Accumulating Dechloromonas spp. Predominating in a Full-Scale Oxidation Ditch Wastewater Treatment Plant

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Protocol publication

[…] Microbial genomic DNA was extracted from sludge samples using the FAST DNA Spin Kit for soil (MP Biomedicals, Santa Ana, CA, USA) according to the manufacturer’s instructions. The PCR amplification of 16S rRNA gene fragments for clone library analyses was conducted as described previously with a primer set of 27F and U533R (, ). PCR products were purified with a QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany), ligated into the pGEM-T Easy Vector (Promega, Madison, WI, USA), and cloned into E. coli JM109 competent cells (Promega). The sequences of the cloned PCR products were elucidated at the Biomedical Center of TAKARA Bio.The sequences obtained were assigned to phylotypes using BLASTClust () with a cut-off value of 97% sequence identity. Phylotypes were phylogenetically classified with the Classifier program in the Ribosomal Database Project () and were compared to sequences in the GenBank nucleotide sequence database using the BLAST program (). Phylogenetic trees were constructed using the neighbor-joining method () with the program MEGA7 (). Bootstrap resampling was conducted with 1,000 replicates to validate the robustness of the phylogenetic trees. The nucleotide sequence data obtained in this study have been submitted to the GenBank database under Accession Nos. LC145217–LC145287. […]

Pipeline specifications

Software tools BLASTclust, MEGA
Applications Phylogenetics, Protein sequence analysis
Chemicals Carbon, Nitrogen, Phosphorus