Computational protocol: Reactivation of Deep Subsurface Microbial Community in Response to Methane or Methanol Amendment

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Protocol publication

[…] The sequence reads obtained from Ion Torrent sequencing were subjected to quality control using the QIIME-software Version 1.9 () using a minimum quality score of 20, minimum and maximum sequence length of 200 and 600 bp, respectively, maximum primer mismatch of two nucleotides (nt) and maximum homopolymer stretches of eight nt. Adapters, barcodes and primers were removed from the sequence reads, and chimeric sequence reads were removed from the dataset with the USEARCH-algorithm () by de novo detection and through similarity searches against the Greengenes reference dataset (Version gg_13_5) () with bacterial and archaeal sequences. OTUs were picked at 97% sequence homology against the Greengenes database, and de novo OTUs were picked from a randomly subsampled sequence subset that failed the closed-reference OTU-picking stage. Singleton OTUs, i.e., OTUs that were represented by a single sequence, were filtered from the dataset. Taxonomy from domain to species level was assigned to OTUs via representative OTU sequences with the Ribosomal Database Project (RDP) classifier algorithm at a minimum of 80% confidence (). Sequences that did not get any taxonomic assignments were removed from the data sets. Alpha diversity calculations were performed on the absolute OUT abundance data as well as on data normalized to 1000 sequence reads per sample for better comparison between samples. Samples with less than 1000 sequence reads were not normalized.The sequences were deposited in the European Nucleotide Archive (ENA) under accession number PRJEB18131. [...] Alpha-diversity measures (observed OTUs, Chao1 OTU richness and Shannon diversity index) were calculated based on the OTU abundance data outputted by QIIME using the Phyloseq package in R (; ) and visualized using ggplot2. The similarity of the archaeal and bacterial communities between the different treatments was tested by principal coordinate analysis (PCoA) using the Phyloseq package in R. The analysis was performed using the OTU abundance data outputted by QIIME. The Bray–Curtis distance model was used for both analyses. Eigen values for the variance explained by the PCoA dimensions were calculated on 999 permutations using vegan () in R. An UPGMA tree clustering the samples according to similarity in the OTU profiles and a heatmap was calculated using the 1000 most abundant OTUs with the Bray–Curtis similarity model using phyloseq in R. Statistically significant differences in number of observed OTUs, Chao1-estimated richness and Shannon diversity between treatment types (original DNA and RNA, amendments) and between the different depths was analyzed with one-way ANOVA, Tukey’s pairwise test, Kruskal–Wallis and Mann–Whitney pairwise test using the PAST software (). […]

Pipeline specifications

Software tools QIIME, USEARCH, RDP Classifier, phyloseq, Ggplot2, vegan
Databases ENA Greengenes
Applications Miscellaneous, 16S rRNA-seq analysis
Diseases Fractures, Bone
Chemicals Methanol, Carbon, Methane