Scaffold provides the user the option to view GO Annotations associated with proteins. These annotations come from a variety of sources including the NBCI and the UniProt GOA knowledge base. Scaffold comes preconfigured with the ability to add the UniProt All Proteomes and Human Only GOA databases. While the All Proteomes database contains a wealth of information regarding numerous species it is very large and takes a long time to download and index. Often users want the ability to quickly search a smaller subset database that is relevant to the species with which they are working. Scaffold allows users to download and search organism-specific GOA databases that are much smaller and therefore downloaded and index more quickly.
For general information about GOA databases and downloads, here are a couple resources:
https://www.ebi.ac.uk/GOA/downloads
http://geneontology.org/page/download-annotations
To download specific databases follow the instructions below:
- Access the UniProt FTP server, also available from the link above.
- From here select the specific organism you are working with and open the corresponding folder, CHICKEN or DOG for example. Note, if you do not see your organism listed on the main page click on the Proteomes folder to bring up a vast collection of organism specific proteomes. You can use your brower's search function to help you locate specific organisms.
- It is important to select the file with the extension GAF.GZ in order for Scaffold to be able to read the data contained therein. Note, select from the standard file or files
*_complex.gaf.gz,*_isoform.gaf.gz, or*_rna.gaf.gzbased on your experimental needs. Consult the UniProt website for more information - Right click the file you are interested in adding and select Copy Link Location
- Follow this pathway: Edit > Edit Annotation Options in Scaffold or Edit > Edit GO Term Options > GO Annotations in Scaffold DDA/DIA. Here you will see any databases that have been added (NCBI is added automatically)
- Select Add or Import Annotations. Here you have the option the add the All Proteomes and Human Only UniProt databases. In Scaffold DDA/DIA you will also see additional options for various organisms
- Select Other Web Site... from the dropdown menu and paste the copied link into the the box. You can also download GAF.GZ files and select Other File... and point this to the path on your local file system
- Give your database a name and choose a folder to save your GOA database to. This should be a local folder that you have permissions to read/write to (where you store your data or FASTA files, for example). Click Add
- Your database should appear under Database Name. Note, the currently selected database is highlighted in green.
- Once your database is selected GO terms can be applied. You can add go terms to an experiment from a selected database by clicking Experiment > Add or Edit Annotations... or Experiment > Apply Annotations in Scaffold DDA/DIA. This dialog allows you to add either GO or Pathway annotations. Choose the appropriate GO term source from the dropdown menu next to the GO Terms button. Then click OK.
- Here you have the option to select the terms displayed, adjust the default and add the selected terms. Simply selecting OK here will apply the default set of GO terms to your experiment file.
- If two or more samples have been loaded, Scaffold has the ability to find enriched terms using the PSEA-Quant algorithm. More information can be found in the User Guide