ApiDB/EuPathDB Workshop

BLAST and Metabolic Pathways Exercises

Omar Harb
Tuesday, June 10th - 9:00 am

Goals of these exercises:

  1. To become comfortable using the BLAST functionality in EupathDB.
  2. To become comfortable using the metabolic pathway functionalities available in EupathDB - ApiCyc and KEGG
  3. Here is a link to a useful resource explaining Blast.

Exercise 1: BLASTing in EupathDB

  1. Begin at the site: www.cryptodb.org
  2. The first thing we will need to do is get a sequence to use for BLAST.
    • Search for the keyword "dihydrofolate". (Hint: under Quick Search on the EupathDB home page).
    • You should get multiple hits. Find the first one that is annotated as "dihydrofolate reductase-thymidylate synthase" (DHFR-TS). (Hint: look in the product description column).
    • Once you find one, click on the gene and go to the gene page. Let's click on the second hit, which is from Cryptosporidium parvum.
    • Scroll down to the bottom of the page for sequence information.
  3. We will start with a protein BLAST.
    • Open a new window and go to the BLAST page from the EupathDB home page. (Hint: under Tools on the EupathDB home page).
    • Copy the amino acid sequence from the bottom of the gene page window from d in step I.
    • Paste the sequence into the input window.
    • Select target data type (let's start with "Proteins").
    • Select BLAST Program. (Hint: BLASTP).
    • Select the target organism (let's start by selecting all).
    • Click on "Get Answer".
  4. Based on the results you should have identified excellent hits in Plasmodium, Toxoplasma and Cryptosporidium. But what about the other organisms in EupathDB? Let's try a different BLAST method.
    • Go back to the BLAST window.
    • Change the target data type to Genome.
    • Select the BLAST Program. Notice you cannot select BLASTP anymore. Try the other options. Notice how your input sequence type has to change when you select a different program. (Hint: TBLASTN is the one you need).
    • Select all target organisms.
    • Click on "Get Answer".
  5. Based on your results you should notice that you now also have sequence from Theileria. However, we are still missing the dihydrofolate from Giardia and Trichomonas. Let's try a different BLAST method.
    • Go to your gene page window and copy the nucleotide coding sequence.
    • Go to the BLAST window and paste the nucleotide sequence into the input window.
    • Select the target data type (try different ones) and the BLAST program. Notice you can only select TBLASTX or BLASTN when your input sequence is nucleotide. (Hint: select TBLASTX).
    • Select the target organisms. This time let's specifically only select Giardia and Trichomonas. (Hint: select the organisms while simultaneously clicking on the apple key on a mac or ctrl key on a PC).
    • Click on "Get Answer".
    • Getting frustrated? Not getting a hit for Giardia and Trichomonas in this case is actually the correct answer! Both of these do not have dihydrofolate reductase or thymidylate synthetase activity.

Exercise 2 : Metabolic pathways in EupathDB

  1. What enzymatic reactions does DHFR-TS participate in?
    • Go to the gene page that you opened in the first step of the last section.
    • Find the CryptoCyc link.
  2. How many enzymatic activities are associated with DHFR-TS?
    • Note DHFR-TS has two entries in CryptoCyc.
    • Click on one of them. Can you find the reactions that are catalyzed by each enzymatic activity?
  3. What about the pathways involving DHFR-TS? Are they viable pathways in Cryptosporidium? Explore the pathways: can Cryptosporidium make PRPP from ribose-5-phosphate? (Hint: click on one of the pathways and explore the next page).
  4. How does the reaction catalyzed by DHFR-TS in Cryptosporidium compare to E. coli?
    • Click on one of the reactions catalyzed by DHFR.
    • On the next page select the "Cross-Species Comparison" tab at the top of the page.
    • Select two or more organisms and click submit.
  5. From the pathways in step III you see that DHFR-TS is involved in de novo biosynthesis of pyrimidines. How can you get a global view of this pathway specifically for Cryptosporidium or other organisms you might be interested in?
    • Go back to the gene page.
    • Find the KEGG pathways and click on one of them.
    • Under pathways on the next page, click on the one that corresponds to pyrimidine biosynthesis.
  6. How does this pathway compare to the same one in Plasmodium falciparum?
    • From the drop down menu select Plasmodium falciparum.
    • Click on go.