Computational protocol: The influence of oxygen and methane on nitrogen fixation in subarctic Sphagnum mosses

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Protocol publication

[…] Sequences from both runs were merged resulting in a total of 216,660 reads with an average length of 252 nucleotides. The reads were analysed using Mothur (v1.38; Schloss et al. ) and the Mothur 454 SOP (Schloss et al. ). First, reads were quality filtered on read length (200–450 bp) allowing for homopolymers (maximum 8), a maximum of 2 differences with the primer sequence and minimum average quality score of 20 over a window of 50 base pairs. Improved reads were aligned to the Silva nr database (release v123) (Quast et al. ). Chimera’s were removed using Uchime. Next, reads were classified at bootstrap value of 80%, after which the unwanted and non-target lineages Archaea, Eukaryota, chloroplast, mitochondria and unknown were removed. The final dataset from which OTUs were clustered consisted of 49,975 sequences of which 16,493 were unique, with an average read length of 221 nucleotides (for a detailed overview of quality filtering see Additional file : Table S3). OTU’s were clustered using the average clustering algorithm at a cut off of 0.03, followed by singleton removal and resulted in 2001 OTUs. Sequences were deposited in NCBI SRA under project number PRJNA432031. [...] Data was analyzed using R version 3.4.0 by the R Development Core Team (). Normality of the residuals was tested using Shapiro–Wilk’s normality test (stats-package). Homogeneity of variance was tested using Levene’s test (car-package). In the analysis of N2 fixation, CH4 oxidation activity and pore water composition, the sites were grouped based on their nutritional trophic state, resulting in the levels: mesotrophic and oligotrophic sites. Grouping was only allowed if the difference between species was not significant (p > 0.05), which was tested first using a one-way ANOVA. N2 fixation activity data was transformed by square root transformation to permit parametric tests. Next, differences in N2 fixation activity under the different O2 conditions, presence of 13C–CH4 and sites (oligotrophic or mesotrophic), were tested using a 3-way ANOVA (stats-package; for sample size (n) see Additional file : Table S5). 13C–CH4 oxidation activity data was normally distributed. Differences in activity between the two oxygen treatments and sites were tested using a 2-way ANOVA (stats-package). All graphs were constructed in R using ggplot2.For downstream analysis of the community composition the OTU table and taxonomy files generated by Mothur, were imported into R and analyzed using phyloseq (McMurdie and Holmes ). […]

Pipeline specifications

Software tools mothur, UCHIME, Ggplot2, phyloseq
Databases SRA
Applications Miscellaneous, 16S rRNA-seq analysis
Organisms Escherichia coli, Bacteria
Chemicals Methane, Nitrogen, Oxygen