Purpose

This is an example workflow that demonstrates how to use CLUSTALW to do a multiple sequence alignment from the command line. It is also to demonstrate how to run this program in non-intractive mode, the first step to programmatic wrapping.
The starting point is DNA sequences

Prerequisites

Access to a linux/unix shell
This work flow assumes that you have the BioPerl libraries and the CLUSTALW binary executables compiled and installed in your path, either by putting them in /usr/local/bin or by editing your $PATH environmental variable.
Note on CLUSTALW

Note that there are many ways to do multiple sequence alignments. This is just one example.

The DNA sequences

I downloaded coding sequences (CDS) for actin genes from five metazoan species from NCBI.
A complete CDS starts at the start codon (ATG; Methionine) and ends at the stop codon (TAG, TGA or TAA).
Codons are three nucleotide units that encode particular amino acids or the stop-translation signal.

This is a sample CDS for C. elegans, in FASTA format:

>c_elegans
ATGTGTGACGACGAGGTTGCCGCTCTTGTTGTAGACAATGGATCCGGAATGTGCAAGGCCGGATTCGCCG
GAGACGACGCTCCACGCGCCGTGTTCCCATCCATTGTCGGAAGACCACGTCATCAAGGAGTCATGGTCGG
TATGGGACAGAAGGACTCGTACGTCGGAGACGAGGCCCAATCCAAGAGAGGTATCCTTACCCTCAAGTAC
CCAATTGAGCACGGTATCGTCACCAACTGGGATGATATGGAGAAGATCTGGCATCACACCTTCTACAATG
AGCTTCGTGTTGCCCCAGAAGAGCACCCAGTCCTCCTCACTGAAGCCCCACTCAATCCAAAGGCTAACCG
TGAAAAGATGACCCAAATCATGTTCGAGACCTTCAACACCCCAGCCATGTATGTCGCCATCCAAGCTGTC
CTCTCCCTCTACGCTTCCGGACGTACCACCGGAGTCGTCCTCGACTCTGGAGATGGTGTCACCCACACCG
TCCCAATCTACGAAGGATATGCCCTCCCACACGCCATCCTCCGTCTTGACTTGGCTGGACGTGATCTTAC
TGATTACCTCATGAAGATCCTTACCGAGCGTGGTTACTCTTTCACCACCACCGCTGAGCGTGAAATCGTC
CGTGACATCAAGGAGAAGCTCTGCTACGTCGCCCTCGACTTCGAGCAAGAAATGGCCACCGCCGCTTCTT
CCTCTTCCCTCGAGAAGTCCTACGAACTTCCTGACGGACAAGTCATCACCGTCGGAAACGAACGTTTCCG
TTGCCCAGAGGCTATGTTCCAGCCATCCTTCTTGGGTATGGAGTCCGCCGGAATCCACGAGACTTCTTAC
AACTCCATCATGAAGTGCGACATTGATATCCGTAAGGACTTGTACGCCAACACTGTTCTTTCCGGAGGAA
CCACCATGTACCCAGGAATTGCTGATCGTATGCAGAAGGAAATCACCGCTCTTGCCCCATCAACCATGAA
GATCAAGATCATCGCCCCACCAGAGCGCAAGTACTCCGTCTGGATCGGAGGATCTATCCTCGCTTCCCTC
TCCACCTTCCAACAGATGTGGATCTCCAAGCAAGAATACGACGAGTCCGGCCCATCCATCGTTCACCGCA
AGTGCTTCTAA

View the whole FASTA file

Doing the multiple sequence alignment with CLUSTALW

CLUSTALW can be run from the command line
It is a binary executable that uses interactive menus

A basic multiple sequence alignment starts with loading the file (select option 1, then enter the filename, actin.fa)

$ clustalw 

 **************************************************************
 ******** CLUSTAL 2.0.9 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:

Then do the alignment by choosing option 2.

****** 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


Your choice:

Then select option 1, and choose the default output file names when prompted. The alignments will be performed and saved to a file as well as printed to the screen.

Enter a name for the CLUSTAL output file  [actin.aln]: 
Start of Pairwise alignments
Aligning...

Sequences (1:2) Aligned. Score:  85
Sequences (1:3) Aligned. Score:  88
Sequences (1:4) Aligned. Score:  90
Sequences (1:5) Aligned. Score:  89
Sequences (2:3) Aligned. Score:  83
Sequences (2:4) Aligned. Score:  86
Sequences (2:5) Aligned. Score:  85
Sequences (3:4) Aligned. Score:  86
Sequences (3:5) Aligned. Score:  86
Sequences (4:5) Aligned. Score:  94
Enter name for new GUIDE TREE           file   [actin.dnd]: 

Guide tree file created:   [actin.dnd]

There are 4 groups
Start of Multiple Alignment

Aligning...
Group 1: Sequences:   2      Score:19741
Group 2: Sequences:   2      Score:20738
Group 3: Sequences:   4      Score:19601
Group 4: Sequences:   5      Score:19209
Alignment Score 74162

Consensus length = 1131 

CLUSTAL-Alignment file created  [actin.aln]


CLUSTAL 2.0.9 multiple sequence alignment


b_xylophilus        ATGTGTGACGAAGAAGTTGCCGCTCTTGTTGTGGACAATGGCTCCGGTATGTGCAAAGCC
p_magellanicus      ATGTGTGACGACGAGGTAGCAGCTTTAGTAGTAGACAATGGCTCCGGTATGTGCAAGGCC
c_elegans           ATGTGTGACGACGAGGTTGCCGCTCTTGTTGTAGACAATGGATCCGGAATGTGCAAGGCC
c_briggsae          ATGTGTGACGACGAGGTTGCAGCTCTCGTAGTGGACAATGGCTCCGGAATGTGCAAGGCC
c_oncophora         ATGTGTGACGACGAGGTTGCTGCTCTTGTGGTTGACAATGGATCCGGAATGTGCAAAGCC
                    *********** ** ** ** *** * ** ** ******** ***** ******** ***

b_xylophilus        GGTTTCGCCGGAGATGATGCCCCACGTGCCGTCTTCCCCTCCATTGTCGGAAGACCCCGT
p_magellanicus      GGGTTCGCCGGAGACGATGCTCCACGCGCTGTGTTCCCCTCCATTGTTGGAAGGCCCCGT
c_elegans           GGATTCGCCGGAGACGACGCTCCACGCGCCGTGTTCCCATCCATTGTCGGAAGACCACGT
c_briggsae          GGATTTGCCGGAGACGATGCTCCACGCGCCGTCTTCCCATCCATCGTTGGACGCCCAAGA
c_oncophora         GGATTTGCCGGAGATGACGCTCCTCGAGCTGTCTTCCCCTCCATCGTCGGCCGACCCCGT
                    ** ** ******** ** ** ** ** ** ** ***** ***** ** **  * **  * 

b_xylophilus        CATCAAGGTGTCATGGTCGGTATGGGACAGAAGGACTCCTATGTCGGAGACGAGGCCCAG
p_magellanicus      CACCAGGGTGTCATGGTTGGTATGGGTCAGAAAGACAGCTACGTAGGAGATGAAGCTCAG
c_elegans           CATCAAGGAGTCATGGTCGGTATGGGACAGAAGGACTCGTACGTCGGAGACGAGGCCCAA
c_briggsae          CATCAAGGAGTCATGGTCGGTATGGGACAGAAGGACTCGTACGTCGGAGACGAGGCTCAA
c_oncophora         CACCAGGGTGTCATGGTTGGTATGGGACAGAAGGACTCGTACGTAGGAGACGAGGCTCAG

Press [RETURN] to continue or  X  to stop:

You are done, the alignment file is named actin.aln

Using CLUSTALW non-interactively

A menu driven-interface is not useful for pipeline or programatic access.
Fortunately, we can run the application by passing the commands via STDIN
This is accomplished by creating a text file with the sequence of commands in it.
```
1
actin.fa
2
1
actin.aln
actin.dnd
X
X
X
```

Annotated version:
- select menu option one, load the input file
```
1
actin.fa
```
- select option 2 (multiple alignments); option 1 runs the alignment.
```
2
1
```
- provide output file names for the alignments and guide tree files
```
actin.aln
actin.dnd
```
- exit from alignment display; alignment menu; main menu
```
X
X
X
```

To run the program non-interactively, save the commands as clustalw_commands.txt, then run CLUSTALW using this incantation:
```
$ clustalw <clustalw_commands.txt
```
program output will scroll rapidly on screen and also save the multiple sequence alignments in actin.aln

Other ways to access CLUSTALW non-interactively

Assembling low-level commands into a file can be tedious
There are pre-rolled wrappers available, such as BioPerl and BioJava.
See an example of how to do the above alignments using BioPerl

iPToL

CLUSTALW_Workflow_Example

Navigate space