Computational protocol: Investigations of potential microbial methanogenic and carbon monoxide utilization pathways in ultra-basic reducing springs associated with present-day continental serpentinization: the Tablelands, NL, CAN

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Protocol publication

[…] Sequences of bacterial 16S rRNA genes from WHC2 pool samples collected in 2010 and 2011 have been previously published (Brazelton et al., ). For this study, additional environmental 16S rRNA gene sequences were collected from the WHC2 pool before and after it was emptied several times and allowed to refill. Filtering of the fluids and DNA extractions of the filters were conducted as described previously (Brazelton et al., ) and briefly summarized here. Sterivex filters were stored on wet ice in the field, frozen in liquid nitrogen as soon as possible, transported on dry ice, and stored at −80°C. DNA extractions were performed by lysis via freeze/thaw cycles and lysozyme/Proteinase K treatment and purified with phenol-chloroform extractions, precipitation in ethanol, and further purification with QiaAmp (Qiagen, Hilden, Germany) columns according to the manufacturer's instructions for purification of genomic DNA. Purified DNA was submitted to the Josephine Bay Paul Center, Marine Biological Laboratory (MBL) at Woods Hole for amplicon sequencing of the bacterial 16S rRNA gene via an Illumina MiSeq platform. Amplification and sequencing protocols developed at the MBL and an updated version of the protocol is available in Nelson et al. (). Quality-filtering of the sequences was conducted via the VAMPS pipeline (Huse et al., ).Previously published environmental 16S rRNA sequences derived from three WHC2 samples collected in 2010 and 2011 (Brazelton et al., ) were generated at the DOE Joint Genome Institute with a MiSeq sequencing platform according to well-established protocols (Caporaso et al., ). The previously published JGI sequences and new sequences reported here from the MBL were generated with similar but non-identical methodologies. Therefore, we also re-submitted two of the 2010–2011 samples for which sequences were previously published (WHC2B-2010 and WHC2C-2011) to the MBL for sequencing with the updated MiSeq protocol to provide a more direct comparison with the 2012 samples.Environmental 16S rRNA sequences were also collected from sediments at the bottom of the pool that were sampled in 2010 and 2011. These sediments were collected from the same location but at a different time than the sediments collected for the 2012 experiments described below. Sediments were sampled by suction with a sterile 60 mL disposable syringe. Sediments were allowed to settle in the syringe for several minutes, and the overlying fluid was expelled. Additional sediment was then sampled by suction into the same syringe, and the settling and fluid expulsion was repeated until the syringe was approximately half-full of sediment. The differentiation between spring- and water column-derived microbes using this method at this site has been previous published by Brazelton et al. (). Sediment samples were contributed to the Earth Microbiome Project (EMP;, which handled the DNA extraction via the MoBio PowerSoil kit and amplicon sequencing via the Illumina MiSeq platform as described by Caporaso et al. ().Taxonomic classification of all sequences was performed using the SILVA reference alignment (SSURefv115) and taxonomy outline (Pruesse et al., ) using the mothur software platform (v.1.32.1) (Schloss et al., ). Figure and supplementary material were generated in R (v.3.1.0) with the phyloseq package (v.1.9.4) (McMurdie and Holmes, ) using the taxonomic counts generated by mothur. Family-level classifications are shown in Figure and supplementary material because the most common sequences could not be classified at lower taxonomic levels. MBL-generated sequences are freely available at the VAMPS database ( under the project code DCO_BRZ. All EMP-generated sequences are available via their database at under EMP Project ID 713: Serpentinite Seeps. […]

Pipeline specifications

Software tools VAMPS, mothur, phyloseq
Databases EMP
Application Metagenomic sequencing analysis
Chemicals Carbon, Carbon Monoxide