David Coil edited Methods.md  over 9 years ago

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Genomic DNA was extracted using a Wizard Genomic DNA Purification Kit (Promega). A nearly full-length 16S sequence was amplified using the 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1391R (5'-GACGGGCGGTGTGTRCA-3') "universal" primers. Sanger sequencing was provided by the College of Biological Science UC-DNA Sequencing Facility (UC Davis). This DNA was also used for Illumina sequencing of the draft genome as described elsewhere (Coil and Eisen, submitted). The genome sequence was annotated using the RAST server \cite{Aziz_2008} \cite{Overbeek_2013}.  A phylogentic tree was inferred using all type strains from the _Erythrobacteraceae_ family. The 1482bp 16S rDNA sequence was obtained from the genome assembly in RAST, and uploaded to the Ribosomal Database Project (RDP) \cite{Cole_2013}. RDP was used to build an alignment including every type strain within the _Erythrobacteraceae_ family, including strain Coronado(T). Because the taxon names exported with this alignment contained special characters that were not compatible with phylogenetic reconstruction software, a custom script was used to remove or replace those characters with underscores. A description of and link to this script can be found in????. in\cite{swabs14}.  FastTree \cite{19377059} was used with default settings to build an unrooted phylogeny. Dendroscope 3 \cite{22780991} was used to view and edit the phylogenetic tree in order to 1) root the tree with a clade that is sister to a well-supported clade containing strain Coronado(T), 2) prune the tree to remove taxa outside the clade containg the outgroup, and 3) "prettify" the tree.