The following examples were shown in the HymenopteraMine Tutorial presented on June 16, 2020, which is available for viewing on YouTube.
A. List Tool. In this example, the HymenopteraMine List Tool is used to upload gene lists and perform gene set enrichment analysis. The data is from: Ma R, Rangel J, Grozinger CM. Honey bee (Apis mellifera) larval pheromones may regulate gene expression related to foraging task specialization. BMC Genomics. 2019;20(1):592. Published 2019 Jul 19. doi:10.1186/s12864-019-5923-7.
In this example, you will upload two gene lists: a background gene list for gene set enrichment and a list of differentially expressed genes. These lists are available either by downloading Additional File 2 from the journal website, or by downloading each of the the formatted text files below. Right click the file names below (or control-click in Mac) to download the txt files.
Apis_mellifera_background_genes.txt – This list was created using the column "background" from Additional File 2 of the publication.
Apis_mellifera_de_genes.txt – This list was created using the column "allDEG" from Additional File 2 of the publication.
You can choose whether to upload the txt files themselves or to open the files in Excel and copy gene ids from a spreadsheet.
- Be sure to login to HymenopteraMine.
- Click the List Tool tab in the navigation bar. If the Upload menu is not showing, click “Upload” in the gray bar just below the navigation bar.
- Select “Gene” as Data Type and “A. mellifera” as Organism.
- Paste the genes from Apis_mellifera_background_genes.txt into the text entry box or click “Choose File” to upload the txt file.
- Click Create List.
- After a database lookup is performed, the number of ids entered and ids found will be provided.
- Enter a name for the list, then click “Save a List of ….. Genes”.
- After the list has been saved, click “View” in the brown bar below the navigation bar and notice that the new list shows up in your list of lists.
- Click “Upload” in the brown bar below the navigation bar, and repeat steps 1 through 7 with genes from the Apis_mellifera_de_genes.txt file, using a new name when saving the list.
- While still on the List Analysis page, scroll down until you see the gene set enrichment widgets. Wait a few moments for the enrichment analyses to complete.
- For the GO Enrichment Widget, change the background gene list by clicking “Change” under “Background Population”, and use the pulldown menu to select your background gene list. If desired, you can change the test correction and maximum p-value. You can use the Ontology pulldown menu to change the ontology from “Biological Process” to “Molecular Function” or “Cellular Component”. Once the analysis is complete, you can download the results by clicking "Download". Results for each ontology must be downloaded separately.
- For the Pathway enrichment widget, use methods described in step 11 to change the background gene list and download the results.
B. Regions Search. In this example, the HymenopteraMine Regions Search Tool is used to search for genes within a list of regions from the A. mellifera genome. Download the following file by right clicking the file name (or control-clicking in Mac).
Apis_mellifera_regions_example.txt – this is a tab delimited file with chromosome id, start coordinate and end coordinate.
- Be sure you are logged into HymenopteraMine.
- Click on the Regions tab in the navigation bar.
- In the Regions Search upload menu, select the organism “A. mellifera”.
- Click the box next to “Select Feature Types” to deselect all the feature types, then select the box next to “Gene”.
- Either paste the coordinates from an Excel spreadsheet into the text box or click “Choose” to upload the txt file.
- For this example, we will not extend the regions or select a strand-specific search (numbers 5 and 6 in the upload menu).
- Click “Search”.
- The search may take a few moments. Once complete, you will see an intermediate result page that will allow you to download results from individual regions or all regions together in various formats. Notice that both official gene set OGSv3.2 and RefSeq genes show up in the search.
- To save the list of all the genes, click “Go” next to the menu to the right of “Create list by feature type”, above the search result table. The pulldown menu provides only “Gene” as a choice, because the search was limited to genes.
- Saving the list brings you to the List Analysis page, as already seen in Example A. The Regions Search tool does not allow you to name the list, but assigns a name like “all_regions_Gene_list_1”.
- Next you will prepare lists to perform gene set enrichment. Since the regions search resulted in genes from two different gene sets, it is necessary to create a new list for each gene set separately. Click the histogram icon within the Gene Source column to enable filtering for individual gene sets.
- A box will appear showing the number of genes in each gene set. First click the box next to “amel_OGSv3.2”, then click the arrow next to “Filter” and select “Restrict table to matching rows”. Then click “Save as a List”, above and to the right side of the table, and select “Gene (1969 Genes)”. This will open a box that will allow you to name a new list and save it by clicking “Create List”.
- Once the OGSv3.2 genes have been saved, use the “Undo” button to undo the previous filtering step. Repeat the filtering step (steps 11 and 12), but this time restrict the rows only to RefSeq genes.
- Now your gene lists are ready for viewing enrichment. The enrichment results showing in the current page are incorrect, because they are based on two gene sets together, even after restricting the table to one gene set. To re-perform the enrichment analysis with the new individual gene sets, click “View” in the brown bar below the navigation bar to view your list of lists. Clicking either one of your new lists will prompt new enrichment computations based on that list. Use instructions from Example A to change the background gene lists in the enrichment widgets to either “A. mellifera RefSeq All Genes” or “A. mellifera amel_OGSv3.2 All Genes”, depending on which list you selected. Repeat this step for the other gene set. Keep in mind that KEGG enrichment is available only for RefSeq genes. Also, note that results of GO enrichment may differ between gene sets due to differences in GO annotations between gene sets.
Please contact us if you have questions about these examples.