1) Constructing the database

Our first step is to construct a database for Primrose to use.  Our database should contain representatives of the target taxon we wish to design a probe for, as well as members of bacterial taxa we want to exclude from the probe's range.  Ideally our database should be as comprehensive as possible without being so big as to impact adversely on the program's performance.  In this example, for illustrative purposes, we use sequence data from a range of sources.

We click 'Construct Database' button and the following dialog box appears:



We next select 'Load Sequence File(s)' and use the resulting Open File Dialog box to load the sequence files which will make up our final database.  Our target taxon is represented by a Fasta formatted collection of 16S rRNA partial sequences, and we load this first:



From a previous analysis we already know our taxon appears to be a novel group within the Bacteroidetes phylum, so next we visit the Ribosomal Database Project and download the sequences for all Bacteroidetes isolates greater than or equal to 1200 nucleotides in length from their preview site (http://rdp.cme.msu.edu/html/analyses_preview.html).  We select to download the data in GenBank format with common gaps removed (to retain their alignment but exclude superfluous gap characters).  The resulting 1304 sequence file is incorporated into our database:



Finally we decide to include small-subunit ribosomal RNA prokaryote data from the Ribosomal Database project Release 8.1 database (included with Primrose within the 'databases' directory).  This database is useful here because (i) it includes a phylogenetic tree which can be used by Primrose, and (ii) it covers a wide range of prokaryote taxa including Archaeal groups.  The prokaryotes data is loaded by selecting the SSU_Prok.phylo file within the 'databases' directory:



The final database appears as follows:




Our database complete, we now press the 'Accept' button and return to the main window.

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