CPGR Workshop – Tutorials Tutorial 1 – Evaluating loci to be ...

CPGR Workshop – Tutorials
Tutorial 1 – Evaluating loci to be candidate diagnostic markers.
Background: A paper from several years ago compared the fully sequenced
Xanthomonas genomes that were available at the time and found unique loci
from each genome. These unique loci would make excellent candidates for
diagnostic markers to detect individual pathovars using PCR. Since the paper
was published, several more Xanthomonas genomes have been sequenced.
Before developing the diagnostic markers, we want to make sure the loci are still
good candidates.
The GenBank Accessions are: AAW77501, BAE69434, AAM38926.
Summary of workflow:
• Search the locus accession in GenBank.
• Examine the GenBank record to confirm the species/pathovar, and
examine fields of interest: locus name, function, etc
• Display the nucleotide sequence of the gene in FASTA format.
• Use the NCBI BLAST server to search all the Xanthomonas sequences in
the non-redundant (nr) nucleotide database at a low expect value (1e-5).
• Interpret the BLAST report, to determine if the locus sequence is unique
and therefore still a valid candidate.
• Run the search again using the same settings as before on the NCBI
BLAST server. However, change the expect value to 10.
• Interpret the BLAST report. Note any differences in the BLAST report.
Detailed Instructions:
1. First, the nucleotide sequence of the locus must be obtained from Genbank at
NCBI. Open the NCBI website in your browser and type the accession in the
search box at the top of the web page.
2. The results page is from Entrez, a tool from NCBI that searches across all the
NCBI databases. You should see the results as shown in the screenshot below:
1 match in the Protein database and 1 match in the Gene Database. Both
databases will take you to the protein sequence. In this case, we’ll look at the
Protein database entry (but feel free to look at the Gene database record for the
locus). Click the Protein: sequence database on the Entrez results page.

3. This displays a summary of the locus record in the protein database.
Click the accession number to display the Genbank record for the locus.
4. Take a look at the general features of the GenBank record.
Can you identify the organism, function, and locus name?
5. Currently we have the protein GenBank record for the gene. However, we
need the nucleotide coding sequence for the gene to carry out the analysis. To
obtain this, scroll down to the FEATURES section of the Genbank record. You
should see a link called ‘CDS’ (coding sequence or ORF). Look at the features
of the CDS. Can you identify the gene name, the genomic location, and the
method the CDS was identified? Now, click on the CDS link to obtain the
nucleotide sequence of the locus.

6. Now you have a GenBank record for the nucleotide sequence of the gene.
Take a moment to look at the record. Can you tell what strand the locus is on in
the genomic sequence?
7. Now the GenBank record has to be converted to the FASTA format in order to
be accepted by the web tools we will be using, such as the BLAST server. At the
top left hand corner of the page, there is a drop down box labeled Display:. Click
the drop down box and select FASTA.

8. You should now have the nucleotide sequence of the locus in FASTA format
displayed in the browser.
9. When doing a bioinformatics analysis, it is useful to keep of log of downloaded
sequences, results, and output in a text editor. This prevents losing work if a
browser crashes or you want to save your work. To do this, open notepad in
windows (Start -> Run – type notepad in the “Open:” box). The FASTA sequence
can be copied and pasted straight from the browser into notepad.

10. Now that we have the sequence of the locus in FASTA format, we can use
the NCBI BLAST server to search the locus against all the Xanthomonas
sequences in GenBank. Go back to the CPGR Workshop Links page and open
the NCBI BLAST Page in the browser. To begin, paste the sequence in the text
box. The locus sequence is the query sequence and will be used to search
against the database of non-redundant GenBank sequences. The sequences in
the database are also known as the subject.
11. Searching against the entire non-redundant GenBank set of sequences is too
wide in this case as we want to limit the search to all of the Xanthomonas
sequences in GenBank. To do this, scroll down to the Choose Search Set panel
and choose the ‘Others’ Database option. Next, select the ‘Custom’ option for the
Organism. Start typing Xanthomonas in the text box. As you type, a drop drown
box will appear showing the available options as your typing narrows them down.
When you see Xanathomonas (taxid:338) appear, select it.

12. Now we have to choose the correct BLAST program. We will discuss this in
more detail tomorrow. For now choose blastn, which searches a nucleotide
sequence against a nucleotide database of sequences.
13. Set the expect threshold to 1e-5. In this case, I also set the max target
sequences to display at 10 to prevent a large amount of data coming back from
the BLAST server.

14. Click the large ‘BLAST’ button to start the search. In a minute or so you
should get a BLAST results report loaded into the browser. A portion of which is
shown below. You can also copy and paste the BLAST report into notepad and
save it. You may want to refer to the BLAST report during the lecture tomorrow.
15. Although we will cover the blast report in detail tomorrow, you should be able
to determine if the locus is unique to the Xanthomonas pathovar? Is the locus a
good candidate for marker development. Why or why not?
16. Now, press the back button on the browser to go back to the BLAST input
page. Change the expect threshold to 10 and run the search again. Does the
BLAST report change? Record your observations for discussion tomorrow.