Computational protocol: Speciationand Reactivity of Uranium Products Formedduring in Situ Bioremediation in a Shallow AlluvialAquifer

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Protocol publication

[…] Genomic DNA was extracted from 5 g of sediment per sample and amplified using universal bacterial 16S rRNA gene primers 27F and 1492R, following methods described by Handley et al. After amplification, Leptospirillum ferrodiazotrophum() 16S rRNA gene amplicons were spiked into samples at 0.5% final concentration to serve as a control. Amplicons were fragmented to an average size of 300 bp, and Illumina libraries were prepared as described by Handley et al. Barcoded libraries were pooled and sequenced on one-sixth of an Illumina HiSeq2000 flow cell (Illumina, Inc., San Diego, CA, USA), and 100 bp long paired-end reads were collected.Reads were trimmed to remove low-quality bases and assembled into (nearly) full-length 16S rRNA gene sequences using the EMIRGE (Expectation Maximization Iterative Reconstruction of Genes from the Environment) method for 16S rRNA gene amplicons. Briefly, 1 million randomly sampled reads per sample were reconstructed into full-length 16S rRNA genes over 100 EMIRGE iterations, and after initial read mapping to a de-replicated version of the SILVA 108 16S rRNA database (http://www.arb-silva.de/). During reconstruction, sequences were clustered into operational taxonomic units (OTUs) based on similarities of ≥97%. OTU abundances, calculated on the basis of the number of mapped reads, were normalized to account for varying sequence lengths. To exclude unreliable rare sequences, only OTUs with raw relative abundances ≥0.01% were used in analyses. This resulted in 334–909 OTUs per sample. [...] Representative OTU sequences were analyzed using the Ribosomal Database Project (RDP, release 10) naïve Baysian classifier, in order to assign taxonomy. For comparison, OTUs were BLASTed against the SILVA 16S rRNA database in order to determine sequence identities. Sequences from all samples were aligned together using SSU-ALIGN 0.1 (http://selab.janelia.org/software/ssu-align) and masked to remove inserted columns (which do not contain aligned nucleotides) as well as regions of low alignment confidence, which were determined by the calculation of posterior probabilities based on alignment to a covariance model. An approximately maximum-likelihood phylogenetic tree was generated using FastTree 2.0.1. The tree was rooted to Thermus thermophilus (X07998.1). Sample communities were compared using phylogenetic (tree-based) and OTU abundance information with weighted Fast UniFrac. Data were analyzed with and without normalization, which accounts for potential differences in OTU branch length owing to different rates of organism evolution. […]

Pipeline specifications

Software tools EMIRGE, FastTree, Fast Unifrac
Applications Phylogenetics, Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Dipturus trachyderma
Chemicals Iron, Uranium