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[…] ples were fragmented to ~ 170 bp using a Covaris S220 Focused-ultrasonicator (Covaris Inc. Woburn, MA) and metagenomic libraries were prepared according to the Nugen Ovation® Ultralow Library system protocol (NuGen Technologies, Inc. San Carlos, CA). Prior to sequencing, metagenomic libraries were visualized on an Agilent DNA 1000 Bioanalyzer chip (Agilent Technologies, Santa Clara, CA) and quantified using a KAPA SYBR® FAST Universal qPCR Kit (KAPA Biosystems, Boston, MA). Paired-end sequencing (2 × 113 bp) was performed on an Illumina HiSeq 1000 (Illumina, Inc. San Diego, CA). Base calls, sample demultiplexing, quality scores, and individual FASTQ files for each sample were generated on a CASAVA 1.7 + pipeline (Illumina Inc. San Diego, CA)., Forward and reverse reads from paired-end sequenced DNA libraries were assembled using the FLASH software with default parameters (minimum overlap, 10 nt; maximum allowed ratio between the number of mismatched base pairs and the overlap length, 0.25). Merged paired-end reads where assembled without further filtering with MEGAHIT v0.3.3-a software using 27 to 123 k-mers range in steps of 10, all other parameters set to their default values. Gene prediction on subsequently generated contigs was performed with Prodigal version 2.60, metagenomic mode. The sequence pool thus generated was screened with RefSeq nr (release 68, Nov 3, 2014) and COG database updated in 2014 using USEARCH v6.0.307 software and –ublast/-evalue < = 1e-04 parameters. Normalization of bacterial metagenomic hit counts was performed by comparing with a set of 40 well known single-copy gene families universally distributed among prokaryotic genomes, which are present in the COG database. Determination of cyc2 gene homologs differential expression was performed by direct sequence similarity search of 2 cyc2 homologs namely AKN78226.1 (Cyc2PV-1 from Mariprofundus ferrooxydans) and ADE10507 (Slit_0265 Sideroxydans lithotrophicus), against the appropriate full reads data sets., Elemental concentrations of filtered and acidified groundwater were characterized at low resolution using an Agilent […]

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