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BIRLA INSTITUTE OF SCIENTIFIC RESEARCH, JAIPUR


Day1 Exercise - Session II

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Section - 1

Sequnce Similartity and Alignment: BLAST, FASTA and Clustal Omega

Lab 1: Local Alignment -BLAST

Basic Local Alignment Search Tool (BLAST) is a dynamic programming algorithm for sequence alignment. It is based on smith-waterman algorithm, means on the concept of local alignment. For an alignment blast uses a query sequence which is taken for the alignment and one or more subject seqeunces on which the query sequence is aligned.
Nucleotide Blast Search a nucleotide database using a nucleotide query
protein blast Search protein database using a protein query
blastx Search protein database using a translated nucleotide query
tblastn Search translated nucleotide database using a protein query
tblastx Search translated nucleotide database using a translated nucleotide query
Here we will go for alignment of nucleotide query sequence i.e. Human Insulin (gi: 186429) with complete nucleotide database.

STEPS:

  1. Open BLAST tool
    Link is :http://blast.ncbi.nlm.nih.gov/Blast.cgi

  2. Click on nucleotide blast (blastn).


  3. Enter fasta sequence or GI number or accession number of query sequence into 'Enter Query Sequence'.

  4. You can set further parameters for blast.

  5. The parameters set there are default, if any change is made in these parameters, a light yellow strip is displayed under that parameter.

  6. After setting up all the parameters click on 'BLAST' button to run blast.

  7. This will take to the result page in some time.

Result page of BLAST

Result inculudes graphics summary, descriptions, dot plots, alignment, scores etc.
In graphics summary result is shown as different color showing a range of percent similarity score.Towards black color, less alignment score means less similarity and towards red color more alignment score means more similarity.
The very first red strip below the range strip with numbers below show the query sequence with its length. After that all colored lines show alignment of query sequence with various (subject) sequences available in database.
After that description of subject sequences are given. It includes name of subject sequence, scores, indentity with query, e-value and the link of subject sequence.
After 'Description' alignment of query sequence with various subject sequences are given. It shows base to base alignment with matches, mismatches and gaps as well.

Lab 2: Global Alignment -FASTA

FASTA (pronounced as fast- aye) is an another tool for sequence similarity search analysis. It makes alignment of query protein sequence against protein database's sequences globally. It is based on needleman and wunsch algorithm.
Two another tools of FASTA suit are FASTX and FASTY. These tools are used for the nucleotide query sequences.
Program Name Description Abbreviation
FASTA Scan a protein or DNA sequence library for similar sequences. fasta
FASTX Compare a DNA sequence to a protein sequence database, comparing the translated DNA sequence in forward and reverse frames. fastx
FASTY Compare a DNA sequence to a protein sequence database, comparing the translated DNA sequence in forward and reverse frames. fasty
SSEARCH Compare a protein or DNA sequence to a sequence database using the Smith-Waterman algorithm. ssearch
GGSEARCH Compare a protein or DNA sequence to a sequence database using a global alignment (Needleman-Wunsch) ggsearch
GLSEARCH Compare a protein or DNA sequence to a sequence database with alignments that are global in the query and local in the database sequence (global-local). glsearch
Here we will go for alignment of protein query sequence i.e. Human Insulin (gi: 386828) with UniProt Knowledgebase database (a primary protein database).[Note: This sequence can be opted from NCBI.]

STEPS:

  1. Open page of FASTA tool
    Link is :http://www.ebi.ac.uk/Tools/sss/fasta/

  2. Select protein database, against the alignment will be performed..


  3. Then input the fasta sequence of protein.

  4. There are various parameters given also to make changes in alignment result. You can set them to default.

  5. Now submit the job and get the alignment result.

Result page of FASTA

The result page of FASTA shows various information regarding to alignment.

Summary Table:
In this portion you will get many subject sequences (searched from the selected database) that have been highly aligned to the query sequence. It also has information of subject sequence lenght, alignment score, percent indentities, percent positives (similarity), and e-value.
You can show alignment of query and subject sequecne by clicking on 'Show' button below the alignment in left side division box.

Tools Output:
It shows the same results as summary table but not in graphically form.

Visual Output:
In this part of result you will get alignment of query and subject sequences in form of colored line. Changes in color are defined as percent similarity according to e-value.

Functional Prediction:
In this page you will get the alignment of functional part of query sequence with function part of other proteins. Mainly these fucntional part in protiens are known as secondary structures. Means this is the alignment result of secondary structure elements.

Submission Details:
This page contains all the parameters details submitted for the alignent.

BLAST and FASTA tools are used for pairwise sequence alignment.

Lab3: Clustal Omega

Clustal-Omega is a general purpose multiple sequence alignment (MSA) program for protein and DNA/RNA. It produces high quality MSAs and is capable of handling data-sets of hundreds of thousands of sequences in reasonable time. It uses seeded guide trees and HMM profile-profile techniques to generate alignments. Minimum three sequences are taken for the MSA.

STEPS:

  1. To perform MSA on Clustal Omega open the following link
    http://www.ebi.ac.uk/Tools/msa/clustalo/

  2. Enter sequences into Input Sequences Box.

  3. Default parameters can be set to change the result of alignment.

  4. Click to submit button to view the alignment result. It will take some time to produce the result.

Results of Multiple Sequcnce Alignment (Clustal Omega)

Alignment:
It shows the base per base alignment of each all sequences. Matches, mismatches, gaps are shown their. There are three types of symbols given below the alignment of some bases:

* [asterisk]     It show strong alignment or identical bases in all sequences.
: [colon]       It shows strong alignment of similar class bases.
. [period]       It shows weak alignment of similar class bases.

Result summary:
It provides alignment result in various format. You can download your msa result as text file from here. You can also get phylogenetic tree file from this page.

Phylogenetic tree
It shows phylogeny among sequences and displays tree.

Submission Details:
This page contains all the parameters details submitted for the alignent.

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