Artemis is a free, cross-platform, application for viewing and annotating sequences. It is able to handle large sequence files such as the E. coliO157:H7 genome.
Artemis can be used to find and annotate ORFs, compare GC plots, and many other things. In this brief tutorial we will import the sequence from sequencher, identify and annotate the ORFs in the sequence
To run artemis you need to install the Java Runtime Environment. (Note: This is only the PC version. You can get the GNU/linux version from Sun, the Mac version from Apple, or the PC version from Sun).
You can download Artemis from UTMEM. (Note: This is only the PC version. You can get other versions from the Sanger Center).
After you have downloaded and started Artemis, open the consensus sequence from sequencher. You will see a window like this:
The top panel shows the stop codons in each of the six reading frames. The bottom panel shows the sequence and translation in each of the six reading frames.
Note that no ORFs are currently shown. We can add those by selecting Create -> Mark Empty ORFs. The default minimum ORF size of 100 is usually sufficient. Five potential ORFs will appear:
However, not all of these ORFs are real, as many do not contain initiation codons. There are two ways to prune the ORFs:
The only difference is that Trim to Any will allow a potential ORF to start with a GTG or TTG, which are occasionally alternate start codons.
In this example, it doesn't matter which you choose. You will be displayed a warning saying that some of the features could not be trimmed (they are now selected). These are likely not real ORFs and we can delete them before continuing. Click OK, and you will see that two of the ORFs remain highlighted. From the Edit menu, choose Delete Selected Features and click yes to the warning. Those features are now removed leaving you with an image like this:
At the same time the ORFs that remain now start with an ATG.
To annotate an ORF we come back to our old friend, the BLAST search. Select an ORF by clicking on it and then choose View Feature Amino Acids as FASTA from the View menu. The sequence will appear like this:
You can now copy and paste this sequence into a new BLASTP window. When the results come back, may want to label that ORF with what it is.
To label a feature, select that feature and choose Edit Selected Features from the Edit Menu. Here you can add an annotation top a feature:
The features will appear in the lower window like this:
To Save your work, you have to do a few shenanigans. Start by saving the ORFs, this is usually called ORFs-100+. Then choose Read Features Into from the file menu, and choose the ORFs file. Then you can delete the ORFs+100 from the Entries menu. Finally, you can save the sequence in whatever format you would like. Genbank is usually the best format