Day2
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Section - 1
Homology Searching
Lab 1: Similarity search using BLAST
In This Exercise We will Get Sequences Similar To The Desired protein sequence of "PYRUVATE KINASE in
Methanocaldococcus jannischii DSM 2661". So first download this protein sequence from NCBI homepage
as we have done in previous section.
The sequence comparison we want to make is restricted to
certain specific genomes, to do this we select 3 completely sequenced genomes viz:
- Sulfolobus solfataricus
- Halobacterium salinarum
- Escherichia coli
STEPS:
- First download the Desired protein sequence of "PYRUVATE KINASE in Methanocaldococcus jannischii DSM 2661"
- Go to the Entrez on NCBI by clicking this link http://www.ncbi.nlm.nih.gov/Entrez
- Click on the Genome whole genome sequence
- from the page displayed click on the BLAST hyperlink at the right side of page
- paste the desired protein sequence in the table displayed box.
- In the query we select protein like this
Query: Protein Database: Protein
- Also select check boxes of certain specific genomes named as:
- Sulfolobus solfataricus
- Halobacterium salinarum
- Escherichia coli
- Later press the BLAST button,
- The web paged viewed will have format option, so select options as you required then press the View Report button
- We get results of the BLAST after this operation
- There are many sequences which are similar to the query; we selected the first three sequences for the three selected organisms.
- ref|NP_342465.1| Pyruvate kinase (pyK) [Sulfolobus solfatar... 244 1e-65
- ref|NP_753966.1| Pyruvate kinase I [Escherichia coli CFT073] 238 1e-63
- ref|NP_279422.1| pyruvate kinase; PykA [Halobacterium sp. N... 197 2e-51
- Click on the links and get the sequences in fasta format from the Display , for the organisms.
- Save all the four sequences in same text file for multiple sequence alignment
Lab 2: Similarity search using FASTA
In this exercise search the similar sequence using the FASTA (Fast Alignment) tool, using the same organism, which have used in above exercise.
Follow these STEPS:
- Go to EMBL Toolbox page by clicking on this link:
http://www.ebi.ac.uk/Tools/ - Now click on Similarity and Homology Hyperlink.
- This will open Similarity searching and Homology page, which contains list of tools. Click on Fasta Protein.
- Now select the required options and paste the Protein sequence of the organism at specified place.
- Click on RUN button.
- It will show you the Summary Table, Different options and list of similar sequences.
- First click on CLEAR ALL button, then select check boxes of required sequences and click on SHOW ALIGNMENTS button.
- study the results and try other options also.
Section - 2
SEQUENCE ANALYSIS
[A.] Pairwise Alignment
- Open SIM Alignment tool for protein sequences by following this link http://www.expasy.org/tools/sim-prot.html
- Copy-Paste Sequences at appropriate place in tool. Download any Protien Sequences from ncbi
- Then perform Alignment, by clicking on Submit button.
[B] MULTIPLE SEQUENCE ALIGNMENT using EBI tool
Follow these steps:
- Search the different species of Rhizobium on the google.com
- From all Rhizobium species choose few species for multiple sequence alignment like:
- Rhizobium cellulosilyticum (AM286429)
- Rhizobium daejeonense (AM910856)
- Rhizobium etli (HB764848)
- Rhizobium galegae (EU074168)
- Download Nucleotide Sequences of these species in FASTA format from http://www.ncbi.nlm.nih.gov
- Open the EBI tools by Clicking on this link:
http://www.ebi.ac.uk/Tools/clustalw2/index.html - Paste the all fasta format sequences of Rhizobium in given text area which you have downloaded
- Now click on RUN button
- You will get results, now study these.
- Now repeat this exercise with same sequences, this time change the differnet parameter values, then study the results, how its differs.
[C] MULTIPLE SEQUENCE ALIGNMENT using Biology Workbench
Follow these steps:
- Open Biology Workbench by clicking on this link
http://workbench.sdsc.edu - New users first create account on this site by clicking on Register , just above Enter the Biology Workbench 3.2 heading.
- Log onto Biology Workbench using your username and password by clicking on Enter the Biology Workbench 3.2
- Select Session Tools and create a new session by selecting start new session from dropdown menu, name it like multiple sequence alignments.
- Click on Nucleic acid Tools
- Select Add New Nucleic acid Sequence and copy paste 3-4 Rhizobium Species Nucleotide Sequences in FASTA format only which u have downloaded in earlier section.
- After the sequences are displayed click on Update button, then save them by clicking on Save button
- Now it will show u check boxes in front of uploaded sequences, check mark those sequences whihc u want to use in multiple sequence alignment.
- Select ClustalW- Multiple Sequence Alignment from drop down menu
- perform multiple sequence alignment by clicking on RUN button
- Observe the results for analysis
- Save the results by clicking on Import Alignment option for further use
- select Download Aligned Sequences from drop down list, check mark ClustalW nucleic acid named Checkbox and then click on Download
- It will show you this link Download/View Sequence(s) in Fasta Format
- Click on this link and save the file.
[D] PHYLOGENETIC TREE PREPARATION: Using BIOLOGY WORKBENCH
- First do the Multiple Sequence Alignment then
- select Alignment Tools select the aligned sequences by clicking the checkbox next to the file name.
- Then select either the DRAWTREE or DRAWGRAM from the tool list.
- Now click on RUN button
- You will now be given some options on parameters you can change in your alignment. You can just use the default values and select Submit at the bottom of the page.
- The tree will now appear.
- DRAWTREE draws a rooted tree.
- DRAWGRAM draws an unrooted tree.
- Same way Generate Distance matrix.
- Do Phylogenetic analysis.