13.11.3) (alpha and beta), gentisate 1,2-dioxygenase (EC 1.13.11.4), homogentisate 1,2-dioxygenase (EC 1.13.11.5), protocatechuate 4,5-dioxygenase (EC 1.13.11.8) (alpha and beta), methyl-coenzyme

M reductase (EC 2.8.4.1) (alpha), methane monooxygenase (EC 1.14.13.25) (particulate: pMMO and soluble: sMMO). The metagenome reads were further compared to a protein sequence library for alkane monooxygenase (alkB) on the freely available Bioportal computer service [66]. The reference library for alkB was downloaded from Fungene (Functional gene pipeline & repository) version v6.1 [74], including only sequences with a score (bits saved) of 100 or more from the HMMER Hidden Markov Model search against NCBIs non-redundant protein database. We used blastX against the protein sequences of the enzyme library with a maximum expectation value of check details 10-20[67]. Maximum one alignment was reported. PCA analysis The PCA-plots were created using the vegan library in R [75–77]. The ordination was based on reads assigned at the phylum level in click here MEGAN version 4 (“Not assigned” and “No hits” were excluded)

and to level I SEED subsystems extracted from MG-RAST (“No hits” was excluded) [68, 69]. All metagenome data were given as percent of total reads. Symmetric scaling, for both parameters and sites, was used in the plot. The C646 ic50 geochemical parameters [25] were fitted onto the ordination using the envfit function. The lengths of arrows for the fitted parameters were automatically adjusted to the physical size of the plot, and can therefore not be compared across plots. To account for the different measuring units, all geochemical parameters were normalized by dividing with the standard deviation and subtracting the smallest number from all numbers in each row. Rarefaction analysis Rarefaction analysis was performed in MEGAN version nearly 4 [68, 69]. The MEGAN program uses an LCA-algorithm

to bin reads to taxa based on their blast-hits. This results in a rooted tree. The leaves in this tree are then used as OTUs in the rarefaction analysis. The program randomly chooses 10%, 20% … 100% of the total number of reads as subsets. For each of these random subsets the number of leaves (hit with at least 5 reads (min-support)) was determined. This sub sampling is repeated 20 times for each percentage and then the average value is used for each percentage. The analysis was done for all taxa (including Bacteria, Archaea, Eukaryota, viruses and unclassified sequences) at the genus level, and at the most detailed level (typically species or strain) of the NCBI taxonomy in MEGAN. Comparison of the metagenomes Comparison tables of absolute numbers for different bacterial and archaeal taxonomic (NCBI) levels for the seven metagenomes were extracted from MEGAN [68, 69]. Likewise, comparison tables of absolute numbers of reads assigned to SEED subsystems in the seven metagenomes were extracted from MG-RAST [72, 73].