Computational protocol: Diversity of Metabolically Active Bacteria in Water-Flooded High-Temperature Heavy Oil Reservoir

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Protocol publication

[…] The amplified alkB, 16S rRNA gene and cDNA fragments of 16S rRNA (about 500 bp) were cloned into the plasmid vector pTZ57RT (Fermentas, Lithuania). Clones containing DNA inserts of the expected size (~500 bp) were sequenced using the plasmid primers M13D and M13R. Sequencing was performed on a 3,730 DNA Analyzer sequencer using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, United States).The sequences of the 16S rRNA and alkB gene fragments were homology-searched using the BLAST software and the NCBI GenBank database (http://www.ncbi.nlm.nih.gov). The sequences were edited using the BioEdit software package (http://jwbrown.mbio.ncsu.edu/BioEdit/bioedit.html). The sequence data were aligned using the CLUSTALW v.1.75 software package (Thompson et al., ), with clones having similarities of 97% or above grouped into operational taxonomic units (OTUs) or phylotypes. Detection of the chimeras and checking for the secondary structure anomalies was carried out using the CHIMERA-CHECK program from the Ribosomal Database Project. Six sequences identified as possible chimeras were found. Unrooted phylogenetic trees were constructed using the methods implicated in the TREECONW software package [http://bioc-www.uia.ac.be/u/yvdp/treeconw.html] and the RDP (Ribosomal Database Project) reference sequences (http://rdp.cme.msu.edu). […]

Pipeline specifications

Software tools BioEdit, Clustal W
Application Phylogenetics
Chemicals Carbon, Iron, Nitrogen, Sulfur