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Jenna M. Lang edited methods.tex
over 9 years ago
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The pooled amplicon 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 are aligned and a consensus is computed using FLASH \cite{Magoc_2011} with maximum overlap of 120 and a minimum overlap of 70 (other parameters 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.
\subsection{Microbial Diversity Analyses}
\subsubsection{OTU assignment and quality control}
Sequences for which the forward and reverse reads had enough overlap to be aligned to create a single consensus sequence were clustered against the greengenes
(gg_13_8_otus) (gg\_13\_8\_otus) OTUs clustered at
97% 97\% similarity. All reads that failed to hit the reference database were clustered \emph{de novo}