Genomic DNA was extracted from 0.60 g (total fresh weight; six replicate extractions) of homogenized forest floor samples using a PowerLyzer PowerSoil DNA isolation kit with a PowerLyzer 24 homogenizer (MoBio Laboratories, Carlsbad, CA). To increase the quality and yield of nucleic acids, we modified the manufacturer's protocol by the initial addition of 250 μL phenol:chloroform:isoamyl alcohol (25:24:1; pH 6.7), bead beating at 4000 rpm for 45 s, centrifugation at 4°C; and overnight ethanol precipitation at −20°C. Extracted DNA was purified using a PowerClean DNA Cleanup kit (MoBio). Purified DNA quality was determined using an ND8000 Nanodrop (Thermo Scientific, Waltham, MA, USA) and quantified by Quant-iT PicoGreen (Invitrogen, Carlsbad, CA, USA) on a Synergy HT fluorometer.
Purified environmental DNA was submitted for shotgun sequencing at the University of Michigan DNA sequencing core. Samples were barcoded by plot (n = 24) and pooled in equi-molar concentrations on six lanes of an Illumina Hiseq 2500 (Illumina, San Diego, CA, USA), with 150 base single-end reads. Illumina reads containing adapter sequences were removed using Cutadapt version 1.7.1 (Martin, 2011 (link)). All metagenome sequence data have been deposited and are publically available in MG-RAST (Meyer et al., 2008 (link)) under accession numbers4614815.3 –4614838.3 .
Purified environmental DNA was submitted for shotgun sequencing at the University of Michigan DNA sequencing core. Samples were barcoded by plot (n = 24) and pooled in equi-molar concentrations on six lanes of an Illumina Hiseq 2500 (Illumina, San Diego, CA, USA), with 150 base single-end reads. Illumina reads containing adapter sequences were removed using Cutadapt version 1.7.1 (Martin, 2011 (link)). All metagenome sequence data have been deposited and are publically available in MG-RAST (Meyer et al., 2008 (link)) under accession numbers