Practical lesson1

PAIRWISE AND MULTIPLE SEQUENCE ALIGNMENTS.
SIMILARITY SEARCHES

• Links to Tools and Databases
• Exercise number 1: Aligning two sequences.
• Exercise number 2. Similarity search with BLAST.
• Exercise number 3. Multiple sequence alignment of sequences identified after a BLAST search.
• Exercise number 4. Discussion: What kind of information is obtained from a multiple sequence alignment?
• Exercise number 5. Identification of coding regions using BLAST.
• Link to tutorial (spanish)

Links to tools and databases:

• Aligning two sequences
• Pairwise Alignment Tools at EBI Both Smith & Waterman and Needle & Wunsch
• Lipman and Pearson's Align program
• NCBI bl2seq
• BLAST
• WU-BLAST at EMBL
• WU-BLAST at EBI
• NCBI BLAST
• NCBI Blast-info
• NCBI Blast-tutorial
• NCBI BLAST download
• ClustalW
• ClustalW at EBI
• ClustalW at ch.EMBNET.org
• ClustalW at crick.genes.nig.ac.jp
• ClustalW at NPS@
• ClustalW at GenomeNet
• Downloading ClustalW
• Running ClustalW by command line
• Databases
• Swiss-Prot
• EMBL
• SRS-EBI
• SRS-EMBL
• Multiple Sequence Alignment viewers
• BoxShade (web server)
• Belvu (info and download)
• Pfaat (info and download)
• JalView (info and download)

Exercise number 1.
Aligning two sequences

• Align the two following sequences, using the Pairwise Sequence Alignment Tools at EBI.

>RPE_YEAST
     MVKPIIAPSI LASDFANLGC ECHKVINAGA DWLHIDVMDG HFVPNITLGQ PIVTSLRRSV
     PRPGDASNTE KKPTAFFDCH MMVENPEKWV DDFAKCGADQ FTFHYEATQD PLHLVKLIKS
     KGIKAACAIK PGTSVDVLFE LAPHLDMALV MTVEPGFGGQ KFMEDMMPKV ETLRAKFPHL
     NIQVDGGLGK ETIPKAAKAG ANVIVAGTSV FTAADPHDVI SFMKEEVSKE LRSRDLLD

>RPE_MYCPN
     MLNLVVNREI AFSLLPLLHQ FDRKLLEQFF ADGLRLIHYD VMDHFVDNTV FQGEHLDELQ
     QIGFQVNVHL MVQALEQILP VYLHHQAVKR ISFHVEPFDI PTIKHFIAQI KQAGKQVGLA
     FKFTTPLVNY ERLVQQLDFV TLMSVPPGKG GQAFNSAVFN NLKQAHKYHC SIEIDGGIKL
     DNIHQIQDDV NFIVMGSGFI KLERWQRQQL LKTNQ

• Please make a global alignment (choosing the option "needle") and also a local alignment (choosing the option "water"). What are the differences between the two outputs? Do you think that these two sequences are related?

• Now try to align the two sequnces using different substitution matrices and changing the gap opening and gap extension penalties. Use, for example, BLOSUM62 and BLOSUM40. Do you see any difference? (you can check the pre-computed results here).

• How could we decide which of the alignments obtained is better?

Exercise number 2.
Similarity search with BLAST.

• Use the sequence RPE_YEAST, to make a BLAST search on a protein database.

• For the moment, please use the EMBL or EBI BLAST servers, since it is easier to retrieve the sequences identified by the BLAST search, and we will use them later, in another exercise.

• If you are using the EMBL BLAST server, use the following parameters:

• database=Swiss-Prot (nrdb95 provides more coverage,  but we would obtain too many related sequences, making more complicated the analysis).
• filter=none
• descriptions=250
• alignments=250

• Once the results of the BLAST search have been returned, you can retrieve the sequences that have been identified as similar, by clicking on "Get selected sequences".

• Here you will find pre-computed results for the EMBL BLAST  search.

• By default, those sequences with the best p-values appear checked, but you could select more or less sequences.

• Those selected by default have been saved in this file.

• Now you can try to use the NCBI BLAST server and compare (the EMBL BLAST server uses WU-BLAST, which is different to the original BLAST developed at the NCBI).

• Here you will find pre-computed results for the NCBI BLAST search.

• Now look for RPE_MYCPN in the output of the two BLAST searches using RPE_YEAST as a query. Check the associated e-value (or p-value) Is the similarity between the to sequences significant?

Exercise number 3.
Multiple sequence alignment of sequences identified after a BLAST search.

In this exercise you will make a multiple sequence alignment of the sequences that have been identified in a similarity search with BLAST.

• CREATING A MULTIPLE SEQUENCE ALIGNMENT WITH ClustalW.

• You can use a ClustalW web server, or run the program locally.
• Using a web server:

• Servers

• Clustal at EBI
• Clustal at ch.EMBNET.org
• Clustal at crick.genes.nig.ac.jp
• Clustal at NPS@.

• Leave the parameters as appear by default, but change the output format:

• output format= GCG (or GCG-msf).

• Running the program locally:

• Downloading ClustalW
• Running ClustalW locally

• You should obtain a file like this.

• VISUALIZATION OF MULTIPLE SEQUENCE ALIGNMENTS (MSAs)

• Multiple sequence alignments can be more easily interpreted if the columns in the alignment are coloured following some criteria (for example, the degree of conservation).

• If you are running ClustalW at the web server of the EBI, you will have the option of getting a coloured alignment. You will have also the options of visualizing the alignment with JalView or constructing a tree.

• If you are running ClustalW by command line, you will obtain a text file with the alignment in MSF format. A popular visualization tool that can be used to enhance the look of your MSA is Belvu (download Belvu for Linux). There is no Belvu version for Windows, though.

• The previously obtained MSA would look like this with Belvu .
• To visualize the alignment with Belvu, simply run the following command from a shell console:

• With Belvu, it is possible to manipulate or perform several kinds of analyses with the alignment, as for example, sorting the sequences according to several criteria, removing redundant sequences, or generating a NJ tree from the MSA.

• Other MSA visualization tools, that you could have installed, are Pfaat and JalView, which are available for Windows. In addition, you can use BoxShade, which is available as a web server.

Exercise number 4.
Discussion: What kind of information can be obtained from a Multiple Sequence Alignment?

Exercise number 5.
Identification of coding regions using BLAST

(From an exercise designed by R. Alonso Allende and MJ Gómez)

>human
AGCTTTCTTCTTTTCCCTGTTGCTCAAATAAATAGTGTTCTTTGCTCAAA
CCCCCTTTCCCTCCTCCTTCTGCAATCTCAGCGCCTAGCGAAATCTGTTT
TCTTCATTGTAACCTCAGCTTCACCGCAATTAATTTTTTTTCCCTCTGGT
CACAAGATAATTCCTGACGCCAGTGAGTCTGGAGGTCAGACGAACAGCAA
ATTGGGGAACAAGGCGGCACTAATTCCTTACAAGTTCCTTGAAAAATCTT
TCGCTTAAAAAAAACGGGGGGTGGGGGGAGCTTCTTTGCTGTTCAGGGAT
TTATGCCTCGCGGAGCTGTGGCTCGAACCAGTGTTGGCTAAGGCGGACTG
GCAGGGGCAGGGAAGCTCAAAGATCTGGGGTGCTGCCAGGAAAAAGCAAA
TTCTGGAAGTTAATGGTTTTGAGTGATTTTTAAATCCTTGCTGGCGGAGA
GGCCCGCCTCTCCCCGGTATCAGCGCTTCCTCATTCTTTGAATCCGCGGC
TCCGCGGTCTTCGGCGTCAGACCAGCCGGAGGAAGCCTGTTTGCAATTTA
AGCGGGCTGTGAACGCCCAGGGCCGGCGGGGGCAGGGCCGAGGCGGGCCA
TTTTGAATAAAGAGGCGTGCCTTCCAGGCAGGCTCTATAAGTGACCGCCG
CGGCGAGCGTGCGCGCGTTGCAGGTCACTGTAGCGGACTTCTTTTGGTTT
TCTTTCTCTTTGGGGCACCTCTGGACTCACTCCCCAGCATGAAGGCGCTG
AGCCCGGTGCGCGGCTGCTACGAGGCGGTGTGCTGCCTGTCGGAACGCAG
TCTGGCCATCGCCCGGGGCCGAGGGAAGGGCCCGGCAGCTGAGGAGCCGC
TGAGCTTGCTGGACGACATGAACCACTGCTACTCCCGCCTGCGGGAACTG
GTACCCGGAGTCCCGAGAGGCACTCAGCTTAGCCAGGTGGAAATCCTACA
GCGCGTCATCGACTACATTCTCGACCTGCAGGTAGTCCTGGCCGAGCCAG
CCCCTGGACCCCCTGATGGCCCCCACCTTCCCATCCAGGTAAGCCTCGAA
GTCGGGACAGGGCTGAACACCCAGGCAAGGATGCTGCGGGACCCTCGGAG
CTCCCGATTGCCTCGCGTAACTCTTCCCTCTTTTCCTCTAATCAGACAGC
CGAGCTCGCTCCGGAACTTGTCATCTCCAACGACAAAAGGAGCTTTTGCC
ACTGACTCGGCCGTGTCCTGACACCTCCAGGTGAGTATCTCCTCTCTTGG
AGAGGGAGGTTTAAACGGCAAGTCCTGGAGTTGGCAGACGTTTTGAAAAA
TTGCCACTCACTCGGTTTAGGGAAACTGAGGCCAGAGAGGGACAAGTGAC
TTGCCCATGGTTGCATCAAATGAATGGCAGAGTCAGTTTCCATGTGATGT
GCATTTAAGCCTTAATGCGCCTGGCCCTGCCTCCGCAGTGGCCGAGGTCT
GGCAAGTAGACATGGTCCGACTAAATACAAGTCTTTCTGTTCCATGTTGT
ATAGGAGCTGTCTTCGGCAGCCCCCTCCCAGCTAGTGTCAATTCCAAGTA
GGAGGGGTAGCGCAACGTCCGCCTGTGGTCTTTGGCGCCAACTGGGTGGG
GGCAGCGTGGGGGGCGGAGTTATCAGGCTGGAGGTACAGACCAAGTTTCC
TCCCTGGCGCCGGCCAGTCTGCGGACGGCCCCCGCCTCGGCACGCTCGGC
GGAAACTGACTGCTCCTTGGTCTTCTTTCCTCCCCCGCCCAGAACGCAGG
TGCTGGCGCCCGTTCTGCCTGGGACCCCGGGAACCTCTCCTGCCGGAAGC
CGGACGGCAGGGATGGGCCCCAACTTCGCCCTGCCCACTTGACTTCACCA
AATCCCTTCCTGGAGACTAAACCTGGTGCTCAGGAGCGAAGGACTGTGAA
CTTGTGGCCTGAAGAGCCAGAGCTAGCTCTGGCCACCAGCTGGGCGACGT
CACCCTGCTCCCACCCCACCCCCAAGTTCTAAGGTCTTTTCAGAGCGTGG
AGGTGTGGAAGGAGTGGCTGCTCTCCAAACTATGCCAAGGCGGCGGCAGA
GCTGGTCTTCTGGTCTCCTTGGAGAAAGGTTCTGTTGCCCTGATTTATGA
ACTCTATAATAGAGTATATAGGTTTTGTACCTTTTTTACAGGAAGGTGAC
TTTCTGTAACAATGCGATGTATATTAAACTTTTTATAAAAGTTAACATTT
TGCATAATAAACGATTTTTAAACACTTGTGTATATGATGACACCCGTCTC
CATTAAGTACTAATGATGCTTTCTCGCACATGGCCGAATTTTGGGAGCTT
TGGGAAAGTGAACTTGCTTATTCTACGAGAGGGAAATGAAAAACTGCCTG
GTTGAGAGGGGATGGGGTGGAGAGAGAAGGGTTCATGATGGGAGTCTCAT
GTCCATTGAGGGATGGGTGCAGAGAAAAGTTCTGGCTCTGCCTCATTATT
TCAGAGATGAAACCAGAGACTGGTGCAAGCT
 

• The nucleotide sequence above is a fragment of the sequence of a human chromosome.

• We would like to check whether there are experimental evidences indicating the presence of  transcribed or coding regions.

• To do it, we will use BLAST to search a ESTs database, using the above sequence as query.

• Access the NCBI BLAST server web page, and select  "Nucleotide-nucleotide BLAST [blastn]".

• Copy the human sequence above, and paste it in the "Search" box.

• Choose "est_human" in the "Choose database" menu, and start the search bY clicking on "BLAST!".

• You should obtain a result similar to this.

• Questions:
• Do you have a clear idea of what are ESTs?
• Do you think that the sequence above contains a transcribed or coding sequence?
• If there were a transcribed sequence, could you infer the structure of the gene?
• Can you think of another strategy to test whether the human sequence contains a coding sequence, using BLAST but NOT a search against the ESTs database?