Evidence of Horizontal Gene Transfer (Module Eight) Instructions

Instructions for Module Eight: Horizontal Gene Transfer
Module Eight looks to collect data that can support potential gene transfers between different species and thus the extent to which two or more species are related. To do this, we will use the EBI’s online tool/database T-Coffee, which looks for the relation between different gene sequences. We will also use tools on the JGI site: the Ortholog Neighborhood Viewer and the GC Percentage Map.

Links: T-Coffee ; JGI’s Online Community Instructions for Module Eight

Phylogenetic Tree

To use T-Coffee we first need to find the desired gene’s and potential xenolog(s)’ (xenologs are homologs that display horizontal gene transfer) amino acid (AA) sequence in FASTA format. To do this:
1) Select the gene of interest on the JGI site – this will take you to the “Gene Detail” page for your gene of interest.
2) Click on the “Homolog Display” link at the top of the page (or scroll down to the bottom of the page)
3) Select “Paralogs/Orthologs” from the scroll down menu
4) Now, scroll down to the “Orthologs” section and select the gene(s) that you want to compare to your gene of interest by selecting the box to the left of the gene
5) Once you have selected the gene(s) scroll down the bottom of the page and click “add selections to gene cart”
6) Scroll down to the “Export Genes” subsection of this page and select the “FASTA Amino Acid format” option. Click “Show In Export Format.”
Note: You will want to save these FASTA sequences somewhere or put them in your gene annotation notebook.

Now that you have the FASTA sequences, go to the T-Coffee site and copy/paste the sequences into the search field and click “run.”

Copy/paste the alignment into the gene annotation notebook.

Scroll to the bottom of the page and click “View As Phylogram Tree.” (Note: the number on the right of each branch of the tree corresponds to the “gene oid” of each gene run through the T-Coffee program”

To save the Phylogram Tree into the gene annotation notebook you will need to take a screen shot. For Macs, use Shift + Command (the apple key) + 4 and select the section of the screen that contains the Phylogram Tree. The image file will appear on the desktop – copy/paste this into the gene annotation notebook. For PCs, use the prntscrn button and paste into the gene annotation notebook

To build a Phylogenetic Tree use another program, such as Dendroscope. Put this into the gene annotation notebook

Gene Context

1) From the “Gene Detail” page of the gene of interest, click or scroll to the “Evidence for Function Prediction” subsection and click on the “Ortholog Neighborhood Viewer” link.
2) Use your mouse to scroll over genes near the orthologous region (this will be the same color in each organism) in different organisms to see if there are any similar genes near the orthlogous region. Similar genes adjacent to the orthologous region are supporting evidence for gene transfer.
3) Right click significant Ortholog Neighborhood Regions and “Save As” the images and upload them into the Gene Annotation Notebook

GC Heat Map

1) From the “Gene Detail” page of the gene of interest, click or scroll to the “Evidence for Function Prediction” subsection and click on “Chromosome Viewer Colored by GC Percentage”
2) Mouse/scroll over the gene of interest (it will be marked by a red bar above it) and record the GC% in the Gene Annotation Notebook
3) Record the “Characteristic GC%” of the genome (it is listed at the top and bottom of the page)
4) Also scroll over neighboring genes and note differences/similarities in the GC%

Reference: JGI’s Online Community Instructions for Module Eight