Computational protocol: Dissecting microbial community structure and methane-producing pathways of a full-scale anaerobic reactor digesting activated sludge from wastewater treatment by metagenomic sequencing

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Protocol publication

[…] Unassembled DNA sequences were annotated using the Metagenomics Rapid Annotation (MG-RAST) server (v3.1). MG-RAST not only enables phylogenetic and metabolic reconstructions, but also provides protein similarities analysis, including both function annotation and function classification []. In the present study, 3.0 Gbp DNA dataset (MG-RAST ID: 4536159.3) was used for most of the analysis. Taxonomic profiles were calculated by Best Hit classification at the E-value cutoff of 10−5 with minimum alignment length of 50 bp based on all the annotation source databases used by MG-RAST. The distribution of taxonomic domains, phyla, orders, families and genus for the annotations was analysed in detail. Concerning taxonomic profiles, percentages shown in the study referred to those classified at a certain taxonomic level.Functional profiling was conducted by the gene annotation with SEED Subsystems using Hierarchical classification at E-value cutoff of 10−5 and minimum alignment length of 17 amino acids [,], respectively, in MG-RAST, and visualized using KEGG mapper. Most of the genes were successfully classified into the hierarchical metabolic categories.To investigate gene profile characteristic for the anaerobic microbial community, the total sequencing reads were annotated against the databases of Clusters of Orthologous Groups of proteins (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG, v59) [,] databases using BLASTP (v2.2.21) with the E-value cutoff of 10−5. Detailed analysis of the anaerobic digestion sludge was conducted to count and compare the hit numbers of the sequences of corresponding enzymes subunits in the methanogenesis pathways. The module ‘KEGGviewer’ in MEGAN was used to analyze pathways [,]. Proteins glutathione-independent formaldehyde dehydrogenase (FdhA), hydrogenase subunit A(EchA), formylmethanofuran dehydrogenase subunit A (FmdA), formylmethanofuran-tetrahydromethanopterin N-formyltransferase (FTR), methenyltetrahydromethanopterin cyclohydrolase (MCH), methylenetetrahydromethanopterin dehydrogenase (MTD), coenzyme F420-dependent N5, N10-methenyltetrahydromethanopterin reductase (MER), tetrahydromethanopterin S-methyltransferase (MtrA), [methyl-Co(III) methanol-specific corrinoid protein]:coenzyme M methyltransferase (MtaA), methyl-coenzyme M reductase alpha subunit (McrA), acetate kinase (AckA), acetyl-CoA synthetase (ACSS), phosphate acetyltransferase (PTA), heterodisulfide reductase subunit A (HdrA), acetyl-CoA decarbonylase/synthase complex subunit beta (CdhC) play important roles in recognized methanogenesis pathways, but lack good representative sequences in the eggNOG and KEGG databases at the time of this study. To accurately discover them, BLASTX results were manually analysed through keyword searches based on NCBI-nr annotations, in which genes representing top BLASTX matches were recovered from GenBank. Confirmation of methanogenesis genes was conducted by manually aligning the matched sequences against NCBI-nr database (9 June 2014) using BLAST with E-value cutoff of 10−10. […]

Pipeline specifications

Software tools BLASTP, KEGGViewer, MEGAN, BLASTX
Databases KEGG
Applications Phylogenetics, Metagenomic sequencing analysis
Chemicals Acetyl Coenzyme A, Methane, Phosphates