User guide
Note: This program requires the
Microsoft .NET framework version 2 or later to be installed on your computer.
The Illuminator Reference files program was designed to aid the production of reference
files for the use of Illuminator. While it is possible to create reference files using Illuminator, this program allows the
rapid production of a large number of reference files specific to a region of the genome. To use the program it is necessary
to download and decompress the sequence data for the chromosomes of interest. These can be obtained from
here and decompressed with 7-Zip, a free application
which can be obtained from here. Illuminator Reference Files
extracts the details of each gene's transcript from a text file created by the
CDS project, the annotated file can be obtained from
here.
Data entry
Since the program links each sequence file to the correct chromosome using the files name, it is important not to change the name
of the decompressed files. To enter the sequence data place the decompressed fasta files (chr*.fa) in an empty folder and press the
Chromosomes button (underlined by a red line in figure 1) and selected the folder. Next enter the
positional information for the transcripts identified by the CDS project by pressing the CCDS button
(underlined by a blue line in figure 1) and select the CCDS data file downloaded from the NCBI web site. Finally, to visualise the
positions of the transcripts press the View button (underlined by a black line in figure 1), this opens
the Reference file creation window (figure 2).
Figure 1
Creating reference files
The Reference file creation window displays the location of gene transcripts for a chromosomal region.
The transcripts are arranged as two rows of black rectangles in the Transcripts panel. The upper row
representing transcripts on the forward strand and the lower row showing transcripts on the reverse strand. Where multiple transcripts
are transcribed from a single gene, a minimum set of all the transcribed exons are displayed. Initially the displayed region is set to
the whole of chromosome 1. To change the current chromosome, select the desired chromosome from the Chromosome
list (underlined by the red line in figure 2).
A specific genomic region can be selected by entering the upper and lower sequence coordinates of the region, entering the name
of a gene or by selecting the region using the mouse. Each mode is selecting using the Search class list
(underlined by the blue line in figure 2). Once a specific region is selected, it is possible to expand this region by pressing the
Zoom button (underlined by the black line in figure 2). It is possible to identify a transcript by placing
the mouse cursor over a black rectangle and reading the genes name from the title bar (Shown in the green rectangle in figure 2).
Selecting a region by entering the sequence coordinates
Select the Physical position option for the list (underlined by the red line in figure 3) and enter the
Start point and End point of the region in the text boxes (underlined by the
blue and black lines in figure 3). To select the region press the Go to button the region will then be
delineated by two vertical black lines in the Transcript panel.
To expand the region, press the Zoom button (underlined by the red line in figure 4). This will show
the transcripts in greater detail, with each gene represented by a black rectangle and the genes exons shown as either green (forward
strand) or orange (reverse strand) rectangles.
Selecting a region by entering a gene name
To select a gene by its name, first select the correct chromosome and then choose the Gene name option
form the list (underlined by a red line in figure 5). Enter the name of the gene in the Name text box
(underlined by a blue line in figure 5) and press the Go to button (underlined by a black line in figure 5).
If found, the upper and lower limits of the gene will be shown by two black vertical lines in the Transcripts
panel. Since the gene may be very short compared to the length of the chromosome these lines may appears as a single line.
Pressing the Zoom button (underlined by a red line in figure 6) will expand the selected region to show the
genes transcript (figure 6).
Selecting a region using the mouse cursor
To manually select a region using the mouse, select the Manual option from the list (underlined by a red line
in figure 7). Then place the mouse cursor at the UPPER limit of the region and press the mouse’s right button, next place the cursor
at the LOWER limit of the region and press the mouse's left button. The region will be delimited by two vertical lines in the
Transcript panel (figure 7)
Pressing the Zoom button expands the selected region (figure 8) and the genes in the region can be identified
by placing the mouse cursor each rectangle as described above.
Creating the reference files
Once a region has been entered, pressing the Create button (underlined by a red line in figure 9) prompts
the user to select a folder to save the reference files in. Illuminator Reference Files then generates the
Illuminator reference files for each of the transcripts in the selected region. If a gene is associated
with multiple transcripts a reference file for each transcript is created.
When all the reference files have been created a message is displayed stating that the files have been completed (figure 10). Since
a minority of the transcripts in the CCDS file contain ambiguous information it may not be possible to create reference files for all
the transcripts, if the region contains a transcript that cannot be used to create a reference file, the genes name and transcript ID
is saved to the errorfile.txt file, which is saved in the same folder as the reference files (figure 11).