PSAT User's Manual

6. Comparing multiple genomes based on those with or without Gene Homologs

In this part of the tutorial, you will compare a set of comparison genomes based on whether they contain homologs to genes in F. tularensis subsp. tularensis

1. Select Francisella_tularensis_tularensis as the Reference Genome.


2. Select the other Francisella genomes above this as the Comparison Genomes.


3. Select the analysis method to Compare multiple genomes.


4. Leave the default BLAST alignment score thresholds setting to the default option (e-value<0.1, bit score > 200, % identity > 30%). The tool will look for BLAST hits that meet all of these cutoff values when determining what to consider a homolog in this analysis. The results can change dramatically when choosing different threshold scores.


5. Leave the display option set to the default value to show all hits for each comparison genome. We assume that the top BLAST hit for each genome is most likely to be a valid ortholog for a given query gene. We therefore have this option to by default include only the most pertinent results and to make the output results more manageable to browse. Users may still be interested in seeing the remaining BLAST hits however, and can select this option if desired.


6. Leave the display option set to the default value to only show hits with a homolog clustering score of at least 2. Since the score predicts the number of genes in a syntenic region, a score of 1 essentially indicates an isolated homolog with no synteny. This default option therefore specifies to only show results in which at least 2 genes make up a syntenic region. Users can still view results that consider isolated homologs in the analysis if desired.


7. Select the Submit button to submit the form. The results indicate the number of genes found in each subset count of comparison genomes.
   
   For example, the results indicate that 192 genes had homologs (as defined by the specified BLAST score thresholds and display options) in none of the selected comparison genomes. This set of genes represents genes unique to the reference genome Francisella tularensis SchuS4. Similarly, the results indicate 1111 SchuS4 genes with homologs in all of the selected comparison genomes.


8. Select and click the entry for 59 genes with a homolog in 1 of the comparison genomes. A page will be displayed with the list of the 59 SchuS4 genes that have homologs in a single comparison genome.
   
   The name or locus tag of the gene is displayed, and the name or locus tag of the homolog is displayed in the appropriate comparison genome column.



9. Select any of the entries in this list of genes. The page with homolog details and genomic neighborhood visualization will be displayed for the selected gene and its listed homologs. You may further explore the homology and genomic context including synteny in this page.


10. Go back in your browser to the gene count results page and select a different result, such as for 2 genomes. Notice that the page of results now indicate which 2 of the comparison genomes have a homolog for each SchuS4 gene listed.


11. Go back to the input page to edit settings such as BLAST score thresholds and display options to test varying the constraints used to determine homology. Modifying these settings can greatly change the numbers of genes in each genome count.