Computational protocol: Metagenomics reveals diversity and abundance of meta-cleavage pathways in microbial communities from soil highly contaminated with jet fuel under air-sparging bioremediation

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Protocol publication

[…] For sequencing ends of fosmid clones and transposon insertion sites, fosmid DNA was isolated at conditions inducing high-copy amplification and purified with the QIAGEN Plasmid Mini Kit system (QIAGEN, Hilden, Germany). Primers used for sequencing reations were the pCC1™ forward and reverse sequencing primers (Epicentre®) and the transposon-specific primers KAN-2 FP-1 and KAN-2 RP-1 (Epicentre®). Sequencing of catechol 2,3-dioxygenase gene fragments was performed on PCR products purified with QIAquick PCR Purification Kit with primers used for sequencing reactions being the same as those used in the original PCR.Sequencing was performed with an ABI PRISM BigDye Terminator v1.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and an ABI PRISM 3100 Genetic Analyser (Applied Biosystems). Raw sequence chromatograms from both strands were assembled with Sequencher software version 4.0.5 (Gene Codes Corporation). Assembled contigs were used for DNA and protein similarity searches on GenBank databases performed with blastn and blastp programs of the National Center for Biotechnology Information website (http://ncbi.nlm.nih.gov). Translated protein sequences were aligned with clustalx 1.83 using default values. Phylogenetic trees were constructed with MEGA4 () using the Neighbour-Joining (N-J) algorithm () with p-distance correction and pairwise deletion of gaps and missing data. A total of 100 bootstrap replications were performed to test for branch robustness.The sequences of genes and PCR fragments reported in this study are available under the EMBL/GenBank/DBBJ Accession No. EU555067 to EU555117 and EU884652 to EU884919. […]

Pipeline specifications

Software tools Sequencher, BLASTN, BLASTP, Clustal W, MEGA
Application Phylogenetics
Organisms Escherichia coli, Pseudomonas putida, Ralstonia pickettii