Computational protocol: Metagenomic Analyses Reveal the Involvement of Syntrophic Consortia in Methanol/Electricity Conversion in Microbial Fuel Cells

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[…] DNA was extracted from biofilms formed on the graphite-felt anode (1 cm2) and cathode membrane (1 cm2), and from planktonic cells in the electrolyte (1 ml) using the FAST DNA Spin Kit for Soil (Q-Bio, Carlsbad, CA, USA). PCR amplification of 16S rRNA gene fragments (V1–V3 region) was performed using primers ad-tag-8F and ad-533R, in which the underlined sequences were adaptors added for pyrosequencing and XXXXXX was an arbitrary tag sequence for sample identification . PCR conditions were described elsewhere . Amplicons were purified using a QIAquick PCR Purification Kit (Qiagen, Tokyo, Japan). Amplicons from different samples were mixed at the same concentration (1 ng µl−1 each), and were then subjected to pyrosequencing using a Genome Sequencer FLX system at the Dragon Genomics Center (Mie, Japan). Phylogenetic analyses were conducted using the DDBJ 16S rRNA database (released on Feb. 21, 2012) and the RDP classifier . An operational taxonomic unit (OTU) was defined as a unique sequence or group of sequences with sequence homologies of over 97%. Alignment of sequences and construction of trees were conducted using the MEGA program ver. 5.1. [...] Total genomic DNAs were extracted from pieces of graphite-felt anode (3 cm2 in total area) using the FastDNA SPIN kit for soil. DNA quality was assessed by agarose-gel electrophoresis, spectophotometeric analysis, and Quant-iT dsDNA BR assay kit (Invitrogen, Carlsbad, CA). Approximately 5 µg of quality-checked DNA was used for library construction and subsequent sequence analyses. High-throughput sequencing of the metagenome samples was performed in the Dragon Genomics Center (Mie, Japan) using a quarter of a lane in the HiSeq 2000 sequencing system (Illumina, San Diego, CA) as described elsewhere . Contig assembly gene prediction, and annotation were conducted as described previously . BLASTP in the BLAST+ software was used to align translated protein sequences to the NCBI-NR database (released in February 2013) with an e-value of 10−2 . The resulting BLASTP files were analyzed according to NCBI taxonomy using MEGAN software version 4.70.4 with the following LCA parameters: Min score, 50; top percent, 10.0; Min support, 5 . The KEGGviewer module in MEGAN was used to map the sequences in each sample to KEGG pathways . Program PEMS, which is based on Tetranucleotide BLSOM was used for the taxonomic assignment of metagenome contigs .Nucleotide sequences determined in the present study have been deposited into the DDBJ Sequence Read Archive database (Accession Number: DRA001173). In addition, metagenome sequences will be provided on request. […]

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