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Jenna M. Lang edited methods.tex
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\subsection{16S rDNA amplicon library construction and sequencing}
DNA was extracted from all samples with the MoBio Power Soil kit, according to the manufacturer's instructions, with a final elution of 50uL. The 16S rRNA genes were targeted for amplification via PCR, using primer constructs that consisted of the "universal" bacterial primers 515F and 806R, both modified by the addition of Illumina adaptor and an 8bp barcode sequence. (All primer sequences used for this study are shown in table XXX). The PCR amplification protocol included a 3 minute denaturation step at 94degC, 30 cycles of (30sec at 94degC, 45sec at 55degC, 90sec at 72degC), and a final 10min extension step of 72degC. PCR reactions were visualized on an agarose gel and purified using AMPure Beads (Agilent Technologies, Inc.) Purified PCR products were quantified with the Qubit® 2.0 Fluorometer (Life Technologies) HS dsDNA assay. All samples were pooled in eqiumolar ratios prior to sequencing on the Illumina MiSeq sequencer.
\subsection{Sequence Processing}
The pooled amplicon
data libraries were de-multiplexed using a custom script (available in a GitHub repository at https://github.com/gjospin/scripts/blob/master/Demul\_trim\_prep.pl). This script allows for 1 base pair difference per barcode to accommodate for sequencing errors. Paired reads
were are aligned and a consensus
was is computed using FLASH \cite{Magoc_2011} with maximum overlap of 120 and a minimum overlap of 70 (other parameters
were are left as default). FLASH output is reformatted (as separate FASTA files,) with appropriate naming conventions for QIIME v. 1.8.0 \cite{Caporaso_2010}. The resulting consensus sequences were analyzed using the QIIME pipeline.