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.
