title: DNA-seq of drainfield surface soil metagenome - nosZ & pmoA amplicons
design: Community DNA was extracted from soil samples using the DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) following the manufacturers instructions. The DNA extracted from these 50 samples was used to amplify target gene sections. We amplified nosZ and pmoA gene fragments in separate, single 50-L reactions using primers nosZ1F and nosZ1662R (Throbck et al., 2004; Wigginton et al., 2018), and pmoAA189cgF and pmoAmb661R (Costello and Lidstrom, 1999; Fernndez-Baca et al., 2019), respectively. Primer pairs included a sequencing overhang (forward primer: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG, reverse primer: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG) at the 5 end. Supplemental Material 1 contains primer sequences and PCR conditions for amplifying gene fragments. We examined PCR products on a 1% (w/v) ethidium bromide-stained agarose gel.Amplicons were purified (Agencourt AMpure XP, Beckman Coulter, Indianapolis, IN), quantified (Invitrogen Qubit Flex Fluorometer, Thermo Fisher Scientific, Waltham, MA), indexed, and pooled equimolarly. Sequencing was performed on paired-end reads of 300 bp on an Illumina Miseq Next Generation Sequencer at the University of Rhode Islands Genetic Sequencing Center.
library_name: AHC21