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CBS >> CBS Courses >> Comparative Microbial Genomics Analysis >> Day 4

Day 4: Pan and core genome plots and Blast atlases


Pan and core genome plots

Pan and core genome plots are graphs that display to what extent gene familes are conserved within a set of genomes. Conservation is evaluated by first BLASTing the proteomes of the genomes againt each other. This is done in a certain order, in that for every proteome, it performs a BLAST search against all previous proteomes. The result is a set of numbers specific for that time point that represents the proteome in the order of the input list, showing:

  • Number of new genes
  • Number of new families
  • Size of core genome
  • Size of pan genome
Two genes are considered to belong to the same gene family if the two are more than 50% identical over more than 50% of the length of the longest of the two genes.

We have prepared a script which produces such a pan- and coregenome plot, provided a list of proteomes.The result is a set of numbers specific for that time point that represents the proteome in the order of the input list. The script will accept a number of proteomes (pr1, pr2, .. prN) and perform a BLAST search of each proteome against all the previous:
  • pr2 against pr1
  • pr3 against pr1+pr2
  • pr4 against pr1+pr2+pr3
  • ...
  • prN against pr1+pr2+pr3 ... pr[N-1]
After these searches, the program will derive the number of core and pan proteins for each proteome. The output list will the be redirected into an R-scriptwhich plots all the core/pan values as a function of the proteome number. Just like the BLAST matrix script you tried last week, this script will cache all the BLAST results. In the event you change the order of the input proteins, all BLAST searches must be carried out again.  However, since you last week did a blast matrix, all of these results are still stored, so changing the order should not be a problem this time.

  • First, log in and create a directory for this work. You will also need X to look at the results. See the previous exercise for how to do this. 
    # log in to the computers again, then
    ssh -Y ibiology
    umask 022
    setenv MAKEFILES /home/people/pfh/bin/Makefile
  • Create a directory where this work will be done.
    # Ensure you are in the right place
    cd ~/
    mkdir coregenome
    cd coregenome
  • Create configuration file for this program
    # create config file
    sh ~karinl/scripts/core/coregenome.sh ../data/prodigal > pancoregenomelist.txt
  • Look at this file using nedit (remember, you need to have X activated!)

    The order the organisms are listed in in the file decides the order of the organisms in the plot. The field on the left is the name of the organism, while the protein file for this organism is listed on the right.
    # look at, and maybe edit using nedit
    nedit pancoregenomelist.txt

    Save the file.

  • Run the pan coregenome plot program.

    # run the program.
    perl ~pfh/scripts/coregenome/coregenome pancoregenomelist.txt > pancoregenomeplot.ps

  • Examine the plot:
    # View the plot
    gv pancoregenomeplot.ps
    Look at the plot. Can you tell how many gene families, approximately, your genomes have in common? How many gene families are there in total for your genomes?


Blast atlases

Blast atlases are similar to the genome atlases that you looked at during Day 3, but in addition to showing genomic properties it also shows blast hits to the target genome.

A blast matrix is always made with a reference organism in the 'middle'. All genomic properties that are shown in the atlas relate to this one organism. Next, other organisms that you wish to compare to the reference organism are searched for genes that are similar to those found in the reference organism. These hits are then shown in the atlas as lines where regions in the reference organism have been found to have a match in the searched organism. One lane per searched organism is shown.

Note: the genes in the reference organism are naturally enough shown in the order they are found in the organism. The hits to a gene are shown where the reference gene is, that is, no inference can be made about the location about the matching gene in the searched genomes.

Zoomable web version

These can be found here: Zoomable atlases

In this version, you find and choose your reference organism first, and then add 'BLAST LANES', one for each of the other organisms you wish to display. Then you press 'submit' and wait a bit. Note: this will only work if you have the the latest java version installed.



Getting your files to your computer


You have now several postscript files in your directories that you might want to have on your computer.

If you have a mac, you can use the ps files directly. If you have a windows computer, you need to do a bit of conversion first. Here is what you do:

You use a command called ps2epsi like this:

ps2epsi <filename>ps

You then have a <filename>.epsi file in your directory.

This file needs to be renamed <filename>.eps

mv <filename>.epsi <filename>.eps



You can then transfer this file to your computer.

Transfer


If you have a mac, use something like Fugu.

If you have windows, use something like WinSCP.


Both of these are graphical secure copy programs. Install them, and connect to login.cbs.dtu.dk with your stud-account.
You can then get the ps or eps files to your computer, and you can then insert them into your documents.