TUTORIAL: MULTIPLE ALIGNMENTS
Oct. 12, 2015
|Warning! Most multiple alignment programs will align either DNA or amino acid sequences. However, it's important to know that unless nucleic acid sequences are very closely-related, with few gaps (eg. tRNA, rRNA genes), a reliable multiple alignment (or for that matter, even a pairwise alignment) is almost impossible. The reason is that nucleic acids use a 4-letter alphabet, allowing many equally good alignments to form a set of sequences. In contrast, the 20-letter amino acid alphabet drastically decreases the number of possible alignments, so that an obvious 'correct' alignment is usually possible to find.|
|Select all amino acid sequences and open the Alignment menu, which shows programs related to multiple sequence alignment|
|Choose TCOFFEE, which brings up the menu|
|TCOFFEE sends the alignment to a new blpalign
Choose File --> Save ALL As and save the t_coffee protein alignment as defensin.pro.t_coffee.fsa
Warning!: Do not use guide trees generated by
clustal or other multiple alignment programs for any purpose
eg. phylogenetic analysis of sequence or species
evolution. These trees are based on pairwise
alignments, and therefore do not contain the evolutionary
information found in the gaps that are present in the
completed alignment. Once you have an alignment, you can
then go back and construct a phylogenetic tree.
|Note on TCOFFEE - To speed
up the alignment, TCOFFEE will break up the problem into
many parts, allocating them to new tcoffee jobs. You can see
these jobs using the Unix top or ps commands. Initially,
there will be many tcoffee jobs. As the profiles for
sub-alignments are merged, the number of tcoffee jobs will
decrease, until the final alignment is completed in a single
While the alignments differ, it is hard to say whether one is "better" than the other. Both programs align the motifs containing the Cys residues. While TCOFFEE tends to insert leading gaps which prevent the N-terminal Met residues from aligning, DIALIGN-TX does not insert any gaps in the N-terminus. The order of sequences in the final alignment is different, seeming to imply a disagreement on the presumed phylogenetic relationships of the sequences. However, Neighbor Joining trees constructed from these alignments are identical (not shown).
MAFFT is a
program that implements a wide variety of alignment strategies.
|One of the unique things to note about MAFFT
is that it will automatically choose the best algorithm for
alignment, based on the number of input sequences, unless a
method is specified. The methods cover most of the
situations typically encountered in multiple alignments.
the type of problem will vary is is best to consult the MAFFT
Algorithms page to see which algorithm best applies to
your specific dataset. For relatively small numbers of sequences
(eg. < 200 sequence) methods such as E-INS-i, L-INS-i and
GINS-i construct a guide tree based on pairwise distances and
then traverse the tree until all sequences have been added,
similar to TCOFFEE.The faster FFT--NS-1 and FFT-NS-2 estimate
between-sequence distances based on frequencies of 6-mer
oligonucleotides. Methods also vary depending on whether or not
they refine the initial alignment by iteratively aligning
subsets of aligned sequences, and by whether they recalculate
the guide tree based on the first alignment, and then
repeat the alignment. Obviously, for short numbers of sequences,
the slower more accurate methods are preferred.
MAFFT, choose Alignment --> MAFFT. Output is
sent directly to blpalign. Try comparing the output using the
FFT-NS-2 and FFT-NS-i, which essentially compares a fast
progressive method with a slower iterative method.
MRTRANS by Bill Pearson aligns DNA sequences using the
corresponding amino acid alignment as a guide. Thus, if you have
aligned a set of amino acid sequences, it is straightforward to
generate the corresponding DNA alignment. MRTRANS requires two
files for input, in Pearson/FASTA format: a file containing the
unaligned DNA coding sequences, and the second file containing the
corresponding amino acid sequences, aligned by a program such as
TCOFFEE, MAFFT or DIALIGNX. When we run MRTRANS from blpalign,
blpalign will automatically generate a protein alignment file from
the proteins selected. Thus, all the user needs to do is to select
a file containing the corresponding DNA CDS sequences, unaligned.
The following example illustrates this process.
1) MRTRANS needs the DNA and aligned amino acid sequences to have the same names. During the extraction process, during translation, the names may be modified, so it may be necessary to change names in 'File --> Get Info', before you export to a .wrp file
2) Where two or more copies of a gene are present in a single entry (eg. CAGTHIOGN:CDS1 and CAGTHIOGN:CDS2), it is necessary to give them each unique names so that MRTRANS can distinguish them. Since the CDS extensions will be removed when MRTRANS is run, one solution is to delete CDS but retain the number (eg CAGTHIOGN_1 and CAGTHIOGN_2).
The current biolegato instances implementation of TCOFFEE and DIALIGN-TX usually handle these steps automatically. Nonetheless, if you are having problems with mrtrans, make sure that the names of sequences are the same for both protein and nucleic acid sequences.
Continuing with our earlier example, if you don't still
have the the T_COFFEE alignment in a blpalign window, read it in
to blpalign from defensin.pro.t_coffee.fsa.
Choose Edit --> Select All.
|To run MRTRANS, choose Alignment --> MRTRANS,
and choose the DNA filename.
This is a very easy step to mess up. Make sure to choose the correct file for DNA unaligned DNA coding sequences.
The aligned DNA sequences appear in a new blnalign window:
Alignments can be edited directly in biolegato instances. By
default, only gaps can be edited out, although it is possible to
delete amino acids or nucleotides if protections are changed (Edit --> GetInfo).
Sometimes, it is useful to set several sequences to act
as a group.
First select all of the sequences by name. Click on the topmost sequence, hold the shift key, and click on GMU12150. This will highlight all but ZMA133530. We can make these sequences function as a group by choosing Edit --> Group. The blpalign window will now look like this:
|The sequences that we have
selected all have a '1' at the left of the name field. This
indicates that they are all members of a group labeled 1.
Any edit done on any sequence in the group will now take
effect on all sequences in the group.
Finally, delete the original gap characters using the Backspace key
or the Delete key.
See the online Help in BioLegato for a more in depth
description of how to edit alignments.
In many cases you need an alignment displayed as editable text. This might be true if you wanted to be able to import the alignment into a word-processor or HTML editor for further modification, such as coloring or underlining certain characters. Choosing Alignment --> REFORM will print out an alignment in which amino acids matching the consensus are indicated by dots:
Various features of the output can be changed in the REFORM menu For example, to print ALL amino acids at every position, the REFORM menu would be set as follows:
10 20 30 40 50 60 70
b) JALVIEW - Graphic display and alignment
10 20 30 40 50 60 70
Maxxxkxxa xxxlxmxLxxatxxx xxxxxCxx xsxxfkglcxsxxxCxx
AF112443_Cmarsiyfma flvlamtlfvaygvq gkeiccke ltkpvk cssdplcqk
AF128239_Cmarsiyfma flvlavtlfvangvq gqnnickt tskhfkglcfadskcrk
BOAJ5280_Cmkntvklsligfvmltvlllgetvia qkrkpcys qepd ktcevn rcka
BOAJ5281_Cmkntvklsligfvmltvlllgetvia qkrkpcys qepd ktcevn rcka
CAGTHIOGN_magfskvia tiflmmmlvfatgmv aeartces qshrfkglcfsksncgs
CAGTHIOGN_magfskvia tiflmmmlvfatdmm aeakicea lsgnfkglclssrdcgn
CAGT_CDS1 magfskvva tiflmmllvfatdmm aeakicea lsgnfkglclssrdcgn
GMU12150_Cmsrsvplvs ticvlllllvatemmgptmvaeartces qshrfkgpclsdtncgs
ZMA133530_mr ivymaav mclvlatmss tspsfcqaggcigcprappppsdetcyedlkcsasrchl
Jalview is a feature-rich sequence alignment viewer. It can be
launched by selecting an alignment in blnalign or blpalign, and
choosing 'Alignment --> Jalview'. (Note: since Jalview
also performs multiple alignments, bldna and blprotein also have
options to launch Jalview).
The alignment above is shown using one of several color schemes available. The Hydrophobicity color scheme shows hydrophobic residues in red, hydrophilic residues in blue, and residues of intermediate hydrophobicity in varying shades of purple.
The alignment can be written in paginated form, suitable for printing, by saving to a PostScript file. PostScript is an almost universal printer language understood by virtually all laser printers. An example of PostScript output can be seen in defensin.jalview.ps. Clicking on this link should launch ghostview or a similar PostScript viewer, which can print the file. The file could also be saved and printed to any laser printer (eg. lpr defensin.jalview.ps).
If your browser is not configured to launch a PostScript viewer, you can save the file, and convert it to PDF. Most Unix systems have the ps2pdf command:
would create a file called defensin.jalview.pdf. Clicking on this link should launch a PDF viewer such as Adobe Acrobat or ggv.
Jalview can also do complete alignments from unaligned sequences.
For a full description of the capabilities of Jalview, see $doc/jalview/contents.html.
|Note to VNC users: Jalview, like many Java applications, has color usage issues. For example, running vncserver at 16-bit color depth (eg vncserver -depth 16) will cause the alignment window to appear completely black. Running vncserver at the default color depth of 8 seems to work (eg vncserver)|