Computational protocol: High Bacterial Diversity of Biological Soil Crusts in Water Tracks over Permafrost in the High Arctic Polar Desert

Similar protocols

Protocol publication

[…] Frozen soil cores were sectioned at −20°C to exclude the outer ca. 1 cm of soil material, to avoid potential sampling contaminants, as previously described . Total community DNA was extracted in a sterile laminar flow hood via bead beating lysis using the MoBio PowerMax Soil DNA Isolation Kit (MoBio Laboratories, Inc. Carlsbad, CA) and supplied protocols. After extraction, DNA concentrations were normalized to 1 ng µl-1. The 16S rRNA genes were amplified using primers targeting the V5 and V6 regions as described by Claesson et al. . Eight base pair barcodes for multiplex sequencing were designed using the BARCRAWL software and were added to the reverse primer. Amplification of 16S rRNA genes was performed using reaction concentrations and thermocycling conditions described previously . Briefly, thermocycling consisted of an initial denaturation of 95°C for 3 min, followed by 30 cycles of 95°C for 30 s, 54°C for 30 s, and 72°C for 30 s, cycling was followed by a final extension of 72°C for 10 min. Successful amplification was verified by gel electrophoresis and amplification products were sequenced via 454 FLX Titanium using standard protocols . Each technical PCR replicate represents an individual PCR reaction (including a unique barcode) generated from the sample DNA template. Therefore each site was represented by three independent technical PCR replicates from both inside and outside water tracks. [...] Raw sequences were processed using the mothur software implementation of PyroNoise and selecting sequences with no ambiguous bases and no homopolymers of >8 bp. Sequences were aligned against the SILVA reference alignment as implemented in mothur and sequences that did not align or did not align over the expected region of the alignment were discarded. Potential chimeric sequences were detected with the UCHIME algorithm and identified chimeras were also discarded.Sequences were clustered into operational taxonomic units (OTUs) using average neighbor clustering and diversity statistics (non-parametric Shannon’s diversity, Shannon’s evenness, and Good’s coverage ) were calculated within the mothur package. Statistically significant differences in diversity metrics were assessed using an ANOVA with a Tukey’s HSD mean separation at p<0.05 using the JMP Statistical Discovery Software version 5.1 (SAS, Cary, NC). As PCR replicates were not found to differ significantly, comparisons among the different sites and inside and outside of water track samples were made between averaged technical replicate values. Pearson’s correlations between soil characteristics or between soil characteristics and bacterial relative abundance were performed with the Free Statistics Software .Principal coordinates analysis was determined using OTU membership and abundance. Comparisons based on community composition were determined using the Jaccard index and comparisons based on community structure employed the abundance-based Jaccard index, calculated in the mother software using the 97% sequence identity OTUs.For the determination of the identity of the dominant OTU representative sequences, the datasets were compiled so that the representative OTUs are the numerically abundant OTUs across the technical replicates and sites. The representative sequences were culled from the 97% OTU data using weighted abundance to ensure representative sequences were the most abundant sequence type among each OTU. Representative sequences from the 25 most abundant OTUs were selected for analysis to limit possible inflation of sequence novelty due to rare species or potential sequencing error. Representative sequences were compared to previously described 16S rRNA sequences using a BLASTn query against GenBank . Taxonomic classification of sequence reads was performed using the Ribosomal Database project taxonomy . […]

Pipeline specifications

Software tools XSTK, mothur, PyroNoise, UCHIME, BLASTN
Application 16S rRNA-seq analysis
Organisms Bacteria, Escherichia coli