Quick Start Project Implementation

1. Obtain and save the query and target database files

From NCBI, download the four genome files in FASTA format with predicted protein sequences:

ftp://ftp.ncbi.nih.gov/genomes/Bacteria/ Bacillus_subtilis/NC_000964.faa
ftp://ftp.ncbi.nih.gov/genomes/Bacteria/ Escherichia_coli_K12/NC_000913.faa
ftp://ftp.ncbi.nih.gov/genomes/Bacteria/ Pseudomonas_aeruginosa/NC_002516.faa
ftp://ftp.ncbi.nih.gov/genomes/Bacteria/ Lactococcus_lactis/NC_002662.faa

and save them in a new folder (here we will use 'aaa') in the PyMood projects folder.
If you have problems with downloading, you can find these files in the folder 'L_lactis'in the 'projects' folder. Copy them to the 'aaa' folder.

2. Rename the data files for ease of manipulation

Because names are easier to memorize than numbers, we recommend you save (or rename):
NC_000913.faa as E_coli.fasta
NC_000964.faa as B_subt.fasta
NC_002516.faa as P_aerug.fasta
NC_002662.faa as L_lactis.fasta


3. Use PyMood BLAST Launcher to generate PyMood import files

The next step of this project is to run a BLAST search of L_lactis.fasta against B_subt.fasta, E_coli.fasta and P_aerug.fasta. L_lactis.fasta is our query database file.

Start PyMood Blast Launcher

4. Select the query database

Click 'Select Query'
In the directory 'projects > aaa' select file 'L_lactis.fasta'
Extraction of FASTA headers should start automatically.
As the message 'Query file has been processed' appears, click 'OK'

5. Select the first target database

Click 'Select Target'
Under the directory 'projects > aaa' select file 'B_subt.fasta'
Extraction of FASTA headers should start automatically
Message 'DB file has been processed' will appear, click 'OK'
Stay with the default options in the PyMood BLAST Launcher window
Click 'Blast!'
Message 'You need to format the database...' will appear
Click 'Format'
While BLAST is running you will see the message box 'BLAST currently running...'
It takes about 6 min to finish BLAST on a 1 GHz processor
Wait until message 'BLAST Done' has appeared
Click 'OK' in the 'BLAST Done' window

6. Select the second target database

Click 'Select Target' again
Select file 'E_coli.fasta'
Message 'DB file has been processed' will appear, click 'OK'
Click 'Blast!'
Click 'Format'
BLAST should start (approximate run time is 6 min on a 1 GHz processor)
Click 'OK' in 'BLAST Done' message

7. Select the third target database

Click 'Select Target' again
Select file 'P_aerug.fasta'
Message 'DB file has been processed' will appear, click 'OK'
Click 'Blast!'
Click 'Format'
BLAST should start (approximate run time is 8 min on a 1 GHz processor)
Click 'OK' in 'BLAST Done' message

When the steps described above are completed, PyMood BLAST Launcher has all the necessary input files for PyMood generated.

8. Use PyMood to display database relationships

Click 'Exit' in PyMood BLAST Launcher window
Start PyMood program
In the 'PyMood Main Menu' window click on 'Data File Setup'
A 'Data File Setup' window should appear

9. Associate the BLAST generated input files with specific colors and generate an RGB file

In the 3D viewer, we would like to display the relationship between Lactococcus lactis proteins and the proteins from the target genomes when:
'Red' axis displays similarity to Bacillus subtilis proteins
'Green' axis displays similarity to Escherichia coli proteins
'Blue' axis displays similarity to Pseudomonas aeruginosa proteins

In the 'Data File Setup' window click on the 'Red' button
In the directory 'projects > aaa' select file 'L_lactis_vs_B_subt.blastp.matrix', click 'Open'

In the 'Data File Setup' window click on the 'Green' button
Select 'L_lactis_vs_E_coli.blastp.matrix', click 'Open'

In the 'Data File Setup' window click on the 'Blue' button
Select 'L_lactis_vs_P_aerug.blastp.matrix', click 'Open'

Click on the button 'Generate RGB File'
The program will prompt to 'save it as'
Save the RGB file as L_lactis.rgb

As soon as the 'L_lactis.rgb' file has been generated and saved, close the 'Data File Setup' window

10. Visualizing the RGB file using the 2D and 3D PyMood viewers

The RGB file is used as input file for the 2D and 3D viewers. To launch 2D and 3D viewers click on 'Project From Data File' in the 'PyMood Main Menu' window.
A 'Project From Data File' window should appear
In the window 'Project From Data File' click on 'Open RGB file'
Select 'L_lactis.rgb' file, click 'Open'
Click on the top 'Select' buton to select the Query annotation, select 'L_lactis.annotation' file, click 'Open'
Click on the next 'Select' button to select Red annotation, select 'B_subt.annotation' file, click 'Open'
Click on the next 'Select' button to select Green annotation, select 'E_coli.annotation' file, click 'Open'
Click on the next 'Select' button to select Blue annotation, select 'P_aerug.annotation file', click 'Open'

Stay with the default settings in the Project from Data File window

Click 'Canvas Plot' button, and a colored canvas will appear
Click 'OK' in the 'Canvas Plot Done' window

Arrange the four windows:
PyMood Canvas
2D Object Info
Annotation Display 1
Annotation Display 2
in a way that you can see all of them simultaneously

Minimize (do not close) all other windows on your screen

Point the mouse over any object on the PyMood Canvas and the Annotation Display 1 will display the annotation information
Double-click on a gene, and the annotation info will be displayed statically in the Annotation Display 2 window.

11. Start 2D Plot

Click '2D plot' in the '2D Object Info' window
A new Canvas '2D Plot' will appear, and the Lactococcus lactis proteins will be sorted accordingly with their similarities to the sequences from the target databases.
Click 'OK' in the '2D Plot Done' window.
Use the press-and-drag right mouse button to select groups of genes.
On Mac OS with a single button mouse press 'Command/Apple' key and click-drag over the objects.
Select a group of the lightest squares; those are the most conservative proteins within the analyzed group of bacteria.
The list of the selected genes will be displayed in the 'Annotation Display 2' window.
In the upper left corner, select the most-black square. This will select the genes which are specific (in the limit of our expectation cutoff in the PyMood BLAST Launcher, e-8) to Lactococcus lactis. Their annotation will be displayed in the 'Ann otation Display 2' window.
By selecting the axially positioned genes, you may view only those shared by the query database and one target database. For example, if you select the red axis squares, you will retrieve the list of genes common between Lactoccocus lactis and Bacillus subtilis only.
To explore this project in the 3D space click '3D Plot' in the '2D Object Info' window.
Please find details of PyMood 2D and 3D Viewer usage in the corresponding sections of this help documentation.