Effective Genome size (EGS) and sampling probability The effective genome size (EGS) for each metagenome was estimated according Selleckchem GSK2126458 to the method developed by Raes et al. [64], using the constants a = 18.26, b = 3650 and c = 0.733. A protein reference database containing the 35 single copy COGs in question were downloaded from STRING (9.0) [64, 65]. BlastX was conducted at the freely available Bioportal computer service [66, 67]. Sampling probability of a random universal single copy gene (1000 bases) and expected number of reads detected was calculated according to Beszteri et al. [26]. Taxonomic

annotation The metagenomic reads were taxonomically classified by BlastX against the NCBI non-redundant Protein Database (ncbiP-nr) [67]. The computation was performed at the freely available Bioportal computer service [66]. Maximum expectation-value was set to 10-3, maximum 25 alignments were reported per hit. The BlastX output files were analyzed according to NCBI-taxonomy in the

program MEGAN, version 4 [68, 69] with default LCA-parameters (Min Score: 35, Top Percent: 10.0 and Min Support: 5). All taxa were enabled. The metagenomes were also analyzed for the presence of gene fragments encoding ribosomal RNA’s using the rRNA and tRNA prediction tool of the WebMGA pipeline [70, 71]. An expectation value cut off of 10-20 was used for the predictions. The reads assigned to the 16S rRNA gene were taxonomically classified by BlastN against the SILVA SSU and LSU databases (version 108). An expectation value cut off of 10-5 was used in the blast analyses and maximum Selumetinib mouse 25 alignments were reported. The BlastN output files were CP673451 combined and analyzed in MEGAN version 4 [68, 69] using the silva2ncbi mapping file. To better capture the taxonomic richness in the relatively few reads assigned to the 16S rRNA gene we lowered the min support threshold while the min score threshold was increased to insure good quality of the hits (LCA parameters: min Score: 50, top percent 10 and min support 1). Metabolic annotation The metagenome reads were assigned to SEED subsystems on the

MG-RAST server (version 2.0) [72, 73]. Maximum expectation-value was set to 10-5, minimum alignment length was set to 100 bases. The SEED subsystems at MG-RAST are organized in a hierarchical structure Bumetanide with three levels, which in the remaining text are referred to as levels I, II, and III, where level III is most detailed. We also searched the metagenomes for key genes involved in hydrocarbon degradation at MG-RAST (version 3.1.2). Maximum expectation-value was set to 10-5, minimum alignment length was set to 50 bases. The genes for the following enzymes where searched; Benzoate-CoA ligase (EC 6.2.1.25), benzoate CoA reductase (EC1.3.99.15) (subunits BadD, E, F, G) benzylsuccinate synthase (EC 4.1.99.11), catechol 1,2-dioxygenase (EC 1.13.11.1), catechol 3,4-dioxygenase (EC 1.13.11.