# Clustalw2 Multiple sequence alignment and phylogenetic analysis allow the identification of conserved positions in protein and nucleic acid sequences. This can lead to an appreciation of the evolutionary history of a group of sequences. * The clustal family of programs is commonly used to produce multiple sequence alignments. Other options are available as well: 1. [Muscle](http://www.ebi.ac.uk/Tools/muscle/index.html) 2. [T-Coffee](http://www.ebi.ac.uk/Tools/t-coffee/) * There are 3 different ways to use the clustal programs. 1. Web-based clustalw can be used [HERE](http://www.ebi.ac.uk/Tools/clustalw2/index.html). 2. ClustalX GUI (and also the command-line clustalw executable) is available for download [HERE](ftp://ftp.ebi.ac.uk/pub/software/clustalw2/2.0.12/). [exampledata](http://luria.mit.edu/Bio2Bioinfo/clustal/) 3. Command-line clustalw2 is installed on rous * The major difference between the 3 options is the interface and the calculation of bootstrap values for the tree, which is only available in the command-line and GUI versions. Other details of running the program are the same. For this lesson, we will use the web-based version to align these sequences. * Login to rous and copy the clustal training files to your home directory. ``` cp -r /net/n3/data/Teaching/IAP_2010_day3/clustal . ``` ``` NOTE: -r means "recursive", copy the folder and everything inside it to the new location "." The lack of a trailing "/" on the clustal path ensures that the folder is moved, not just it's contents. ``` * enter the clustal directory: ``` cd clustal ``` * view the raw sequence files: ``` cat *.pep ``` * launch the clustalw application: ``` clustalw2 ``` * The following interactive menu appears, options 1 and 2 will be used in this demonstration. Additional information is available [HERE](http://luria.mit.edu/bio2bioinfo/clustalw_help) ``` ************************************************************** ******** CLUSTAL 2.0.12 Multiple Sequence Alignments ******** ************************************************************** 1. Sequence Input From Disc 2. Multiple Alignments 3. Profile / Structure Alignments 4. Phylogenetic trees S. Execute a system command H. HELP X. EXIT (leave program) Your choice: ``` * Select option 1 to load sequence and specify the file "tiny.pep". Before being returned to the original meny, you should see: ``` Sequences should all be in 1 file. 7 formats accepted: NBRF/PIR, EMBL/SwissProt, Pearson (Fasta), GDE, Clustal, GCG/MSF, RSF. Enter the name of the sequence file : tiny.pep Sequence format is Pearson Sequences assumed to be PROTEIN Sequence 1: zf_12a1_a 28 aa Sequence 2: zf_12a1_b 28 aa Sequence 3: hs_a11 28 aa Sequence 4: hs_22a1 28 aa Sequence 5: zf_a11 28 aa ``` * From the original menu, now select option 2. Multiple Alignments. This activates the Alignment menu: ``` ****** MULTIPLE ALIGNMENT MENU ****** 1. Do complete multiple alignment now Slow/Accurate 2. Produce guide tree file only 3. Do alignment using old guide tree file 4. Toggle Slow/Fast pairwise alignments = SLOW 5. Pairwise alignment parameters 6. Multiple alignment parameters 7. Reset gaps before alignment? = OFF 8. Toggle screen display = ON 9. Output format options I. Iteration = NONE S. Execute a system command H. HELP or press [RETURN] to go back to main menu ``` * All default options are correct except option 9,select this to see the output format menu: ``` ********* Format of Alignment Output ********* F. Toggle FASTA format output = OFF 1. Toggle CLUSTAL format output = ON 2. Toggle NBRF/PIR format output = OFF 3. Toggle GCG/MSF format output = OFF 4. Toggle PHYLIP format output = OFF 5. Toggle NEXUS format output = OFF 6. Toggle GDE format output = OFF 7. Toggle GDE output case = LOWER 8. Toggle CLUSTALW sequence numbers = OFF 9. Toggle output order = ALIGNED 0. Create alignment output file(s) now? T. Toggle parameter output = OFF R. Toggle sequence range numbers = OFF H. HELP ``` * User toggle #4 to activate PHYLIP output, hit return to revisit the alignment menu * Select option 1 to do the alignment. Accept the default conditions and hit return until you end up at the main menu. Exit the program with "X". ``` cat tiny.[ap][lh][ny] ``` ``` NOTE: Square brackets are regular expression syntax. For example [ap] = either an "a" or a "p" at that position. ``` This is the clustal format output: ``` CLUSTAL 2.0.12 multiple sequence alignment zf_12a1_a VADLVFLVDGSWSVGRENFRFIRSFIGA-- zf_12a1_b KADLVFLIDGSWSIGDDSFAKVRQFVFS-- hs_22a1 HYDLVFLLDTSSSVGKEDFEKVRQWVAN-- hs_a11 YMDIVIVLDGSNSIYP--WVEVQHFLINIL zf_a11 YMDIVIVLDGSNSIYP--WNEVQDFLINIL *:*:::* * *:  :  :: :: ``` \ This is the phylip format output: ``` 5 30 zf_12a1_a VADLVFLVDG SWSVGRENFR FIRSFIGA-- zf_12a1_b KADLVFLIDG SWSIGDDSFA KVRQFVFS-- hs_22a1 HYDLVFLLDT SSSVGKEDFE KVRQWVAN-- hs_a11 YMDIVIVLDG SNSIYP--WV EVQHFLINIL zf_a11 YMDIVIVLDG SNSIYP--WN EVQDFLINIL ``` --- # Agent Instructions: Querying This Documentation If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question. 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