Download Case Studies in Bioinformatics

Bioinformatics Worksheet
Biology 22
Directions for Bioinformatics Exercise
Prior to this laboratory, your instructor has prepared sequence files that will allow you to
complete all of the steps of the bioinformatics analysis. You do not need to search for high
quality data, edit any of the posted files/chromatograms, or move any of the
files/chromatograms as described in Part 1 of the Chapter 9 instructions. You will access the
data from Dr. Colavito’s homepage. As you will not be able to edit or alter the posted
information, you should establish your own text or spreadsheet files to keep track of the data
analysis.
To access the sequence data, navigate to Dr. Colavito’s homepage:
http://homepage.smc.edu/colavito_mary/biology22/course.htm
and select the “Bioinformatics Sequences and Search Tools” link:
http://homepage.smc.edu/colavito_mary/biology22/bioinformsequences-W11.htm.
Part A: Determine Sequence Identity Using BLAST (see page 193 of Chapter 9 instructions)
For this section, you need a FASTA formatted file containing four sequences to analyze.
These are already posted on the Bioinformatics Sequences page of Dr. Colavito’s homepage.
Follow the alternate directions for steps 2.1 and 2.2 and then refer to BioRad instructions for
the remaining steps.
2.1 Alternate Directions: Navigate to the Bioinformatics page and select the
sequence assigned with your worksheet.
2.2 Alternate Directions: Save the FASTA formatted sequences in a text file for
future use. (We are not using the iFinch site for data access or storage.)
Follow the Bio-Rad instructions for
2.3 Doing a batch blastn search with your FASTA sequences
NCBI BLAST Link: http://blast.ncbi.nlm.nih.gov/
NOTE: Entering Arabidopsis thaliana as the Organism may be helpful.
2.4 Introduction to FASTA file format
2.5 Understanding blastn results
Part B: Assemble the Sequences (see page 208 of Chapter 9 instructions)
Note: you will not need to “Correct Mistakes in the Base Calls” as mentioned in this section.
Follow the Bio-Rad instructions for
3.2 Assembling the sequences using CAP3
Be sure to save your Contig sequence(s) and Assembly Details results
Part C: BLAST Search on the Contig Sequence (see page 220 of Chapter 9 Instructions)
Follow the Bio-Rad instructions for
4.2 Doing a blastn search on your FASTA-formatted contig sequence
4.3 Analyzing your blastn results
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Bioinformatics Worksheet
Biology 22
Part D: Determine Exon Identity with Genscan (Shortcut for sections 5 and 6 of Bio-Rad
Instructions)
Search Tool Genscan at MIT
Go To
http://genes.mit.edu/GENSCAN.html
Steps to
 Under Organism, choose Arabidopsis
Take
 Keep all other settings as initially shown
 Paste your FASTA-formatted contig sequence into the
white query box
 Click on Run Genscan
 Record the number of exons predicted from the scan,
“suboptimal” exons are acceptable
(Key: Init=Initial, Intr=Internal, Term=Terminal exons)
 Record the number of amino acids in the largest predicted
peptide sequence
 Copy the Genscan output for future use
 Copy the largest predicted peptide sequence from the
results and save it in a text file
Part E: Confirming the Identity of the Predicted Peptide Sequence
(Repeat the basic BLAST search using blastp for protein sequences)
Search Tool
Go To
Steps to
Take
BLAST at NCBI
(Basic Local Alignment Search Tool)
http://blast.ncbi.nlm.nih.gov/
 Under Basic BLAST, Choose protein blast
 Paste the predicted peptide sequence in the query box
 For Database, choose Reference proteins (refseq_protein)
 For Organism, type plants
 For Algorithm, choose blastp (protein-protein BLAST)
 Click on the blue BLAST button at the bottom of the page
 Scroll down the results page to “sequences producing
significant alignments”
 Identify proteins that show a significant homology to the
predicted peptide sequence and record the information on
the summary table
 Reformat the results to produce an alignment file using the
following steps (similar to process described on pages 232233)
 Near the top of the screen, choose Formatting Options
 For Show, choose Plain Text instead of HTML, and check
the Advanced View and Use Old Blast Report Format
boxes
 For Alignment View, choose Query-anchored with dots for
identities
 For Limit Results, change all three numbers to 10
 Choose Reformat in the upper right corner
 Save the resulting information in a text file for submission
with your worksheet.
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