Exercise: Identify an organism through its genes (ORF-Finder, BLASTP)
Imagine that you are a medical researcher in a hospital. Recently, patients with a mysterious and ultimately lethal infection have begun coming in. You need to identify the infectious agent.
Laboratory protocol:
- EM analysis of body fluid samples reveals unusual particles.
- These particles turn out to contain RNA.
- Reverse translating the RNA allows to sequence it.
- Try to determine any potential genes in the sequence by determining whether it contains open reading frames (ORFs).
- Using the genes determine the organism.
Bioinformatics protocol:
- Try to identify genes by finding ORFs.
- Copy and highlight the pathogen's sequence.
- Go to http://www.ncbi.nlm.nih.gov/.
- On the left find and click the link Tools.
- Find and click ORF Finder.
- Paste the sequence below into the window and click OrfFind.
- What do the six horizontal bars mean?
- How many potential ORFs (green boxes) were identified?
- Change the ORF-size from 100 to 300, click Redraw.
- How many ORFs did the tool identify now? In what Reading Frames?
- Clicking on an ORF will allow you to view the nucleotide sequence and the deduced amino acid sequence for this ORF. I t will also relate the ORFs in the graphic to the ORF-listing at the right.
- In order to determine whether any of the ORFs resembles known genes submit their sequences to database searches using BLASTP. BLASTP utilizes the deduced amino acid sequence to search for similar proteins.
- Click an ORF (green box).
- Find Program, set it to blastp.
- Find Database, set it to nr.
- Click BLAST.
- Record the request ID.
- Click Format!.
- Using the output from the BLAST search identify the gene/protein and, if possible, the organism.
- If you can't determine the organism, yet, repeat this procedure for another ORF.