Computational protocol: Temporal dynamics in microbial soil communities at anthrax carcass sites

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Protocol publication

[…] Environmental DNA samples were sequenced at the Norwegian Sequencing Centre (NSC) using Illumina MiSeq® (Illumina Inc., San Diego, California, USA). A 250 bp paired-end sequencing library (400 bp insert length) was generated using a Regular TruSeq® adapter ligation kit (Illumina Inc., San Diego, California, USA). Fastq files were processed using cut-adapt (v1.8) [] and prinseq-lite (v0.20.4) [] (See Additional file : for settings). Metagenomic read GC-content was calculated using infoseq (EMBOSS v. 6.5.7 []). The infoseq output was used to calculate average GC-content and GC-content skewness (timeData package version: 3012.100) using R-Studio (v3.3.0).The average genome size (AGS) of each metagenome was estimated using MicrobeCensus on clean paired-end files (Additional file : Methods). The likelihood of sampling a universal single copy is inversely correlated with the AGS of a metagenomes [, ]. Thus universal single copy abundance will show significant differences when AGS between metagenomes differ. [...] Two methods were used to quantify B. anthracis in the soil samples; serial dilution culturing and quantitative PCR (qPCR) (see Additional file : for details).The genome coverages of the B. anthracis isolate K1 and K2 genomes (Accession numbers: LBBZ00000000 and LBCA00000000 []) isolated from the Ca1 and Ca2 sites respectively, were investigated by mapping metagenomic sequences using the Burrows-Wheeler Aligner (BWA v0.7.8) []. The genomes of B. cereus E33L, B. thuringiensis HD-771 and Bacillus subtilis 168 were also used as references (see Additional file : for details). [...] Taxonomic composition of the metagenomes was determined using metaxa2 (v2.0.1) which extracts SSU rRNA sequences [], MEGAN (v5.10.15) [], Kraken (0.10.5-beta) [] and metaBIT []. For the details on the comparison of the taxonomic classifiers see Additional file . The metaxa2 taxonomic profiles were visualised with a heatmap using ggplot2 in R-studio (see Additional file : for settings). Metaxa2 extracted SSU reads were run through the MetaAmp 1.1 pipeline to obtain operational taxonomical unit (OTU) abundances.Shotgun sequences from each dataset were compared to the NCBI nr database (accessed on 20.01.2016) using Diamond (v0.7.11) [] and the output was used for taxonomic and functional classification in MEGAN (v5.10.15) []. Temporal species composition dynamics of metagenomic samples was examined by principle coordinate analysis (PCoA) in R-Studio (v3.3.0). Changes in the metabolic potential of the community over time were analysed by correlation of KEGG KO-terms and pathways [, ] abundances with AGS size (For details see Additional file ). […]

Pipeline specifications

Software tools PRINSEQ, EMBOSS, MicrobeCensus, BWA, metaxa, MEGAN, Kraken, Ggplot2, MetaAmp
Applications Miscellaneous, Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Bacillus anthracis, Escherichia coli, Bacillus cereus