Computational protocol: The metagenomic approach to characterization of the microbial community shift during the long-term cultivation of anammox-enriched granular sludge

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Protocol publication

[…] Both 16S rRNA amplicons and metagenomes sequencing were performed using the Illumina approach. The sequencing results were recorded as fastq files and uploaded to the Meta Genome Rapid Annotation Subsystems Technology (MG-RAST) server for analysis (Meyer et al. ). Each file was subjected to quality control (QC), which included quality filtering (removing sequences with ≥5 ambiguous base pairs) and length filtering (removing sequences with a length ≥ 2 standard deviations from the mean). The automated pipeline provided by MG-RAST was also used to obtain taxonomic classification using the BLAT program referencing the SILVA database. All the identifications were made using a maximum e-value of 1e-5, a minimum identity cutoff of 90%, and a minimum alignment length of 50 bp. The functional profiling was conducted by gene annotation with SEED subsystems using a hierarchical classification at E-value cutoff of 10–5 and a minimum alignment length of 15 amino acids in MG-RAST. Visualization was carried out using KEGG mapper. Taxonomic and functional differences between metagenomes were analyzed using Statistical Analysis of Metagenomic Profiles (STAMP v. 2.1.3) (Parks and Beiko ). Statistically significant differences between metagenomes were identified by Fisher’s exact test combined with the Newcombe-Wilson method for calculating confidence. The Illumina metagenomic datasets are available at MG-RAST. Sequences data of the 16S rRNA gene are deposited under accession numbers 4629229.3 (AMX_2013), 4629228.3 (AMX_2015), whereas shotgun sequencing data are available under numbers: 4647330.3 (AMX_2013B), 4647329.3 (AMX_2015B). […]

Pipeline specifications

Software tools BLAT, STAMP
Databases MG-RAST
Application Metagenomic sequencing analysis
Chemicals Ammonia, Nitrogen