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TUTORIAL: MULTIPLE ALIGNMENTS

Nov. 18, 2024

Example: Plant Defensins

1. Eliminate redundant sequences from the dataset

Select all amino acid sequences and open the Alignment menu, which shows programs related to multiple sequence alignment

2. Clustal Omega - Global multiple alignment by clustering

Select the non-redundant sequences resulting from CD-HIT, and choose Alignment --> Clustal Omega.

Clustal sends the alignment to a new blpalign window. Choose File --> Save ALL As and save the protein alignment as defensin.pro.cdhit.clustalo.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.

Choose Edit --> Select All, and then Edit --> Get Info. Change the Colour mast to protein_hydrophobicity. (Note: If the colors don't change, either resize the BioLegato window or scroll a bit using one of the scroll bars. This forces BioLegato to redraw the sequence canvas.)

3. MAFFT - Multiple Alignment by Fast Fourier Transformation

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.

Because 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 Clustal Omega.The faster FFT--NS-1 and FFT-NS-2 estimate pairwise sequence distances based on frequencies of 6-mer oligonucleotides or oligopeptides shared between each pair of sequences. 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.

To launch 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.

Weblogo has many options for presentation of the logo. For now, we'll just add a title to the Logo title box. Note that one quirk of Weblogo is that it doesn't allow blank spaces in titles. To separate words, simply use an underscore (_) wherever a blank would normally be used. The title here is "Plant_Defensins"

Weblogo plots information content of each position in the alignment in bits of information. The information content can be thought of as a deviation from randomness. A high bit score would be a highly unlikely outcome, typically occurring where specific amino acids are conserved. A low bit score would mean closer to a random distribution of amino acids at a position, typically at less conserved positions. Note that we can see fairly high bit scores at the K/R positions mentioned earlier. The heights of the letters indicate the relative frequencies of each amino acid, at that position in the alignment.

4. pal2nal.pl - Alignment of DNA sequences, using protein alignment information

pal2nal.pl by Mikita Suyama 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. pal2nal.pl requires two files for input, in 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 Clustal Omega or MAFFT. When we run pal2nal.pl from blpalign, blpalign will generate a DNA 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.
 

Notes:  1) pal2nal.pl 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 .fsa 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 pal2nal.pl can distinguish them. Since the CDS extensions will be removed when pal2nal.pl is run, one solution is to delete CDS but retain the number (eg CAGTHIOGN_1 and CAGTHIOGN_2). The current BioLegato instances implementation of Clustal Omega and MAAFT usually handle these steps automatically. Nonetheless, if you are having problems with pal2nal.pl, make sure that the names of sequences are the same for both protein and nucleic acid sequences. 3) Before running pal2nal.pl, blpalign compares the list of sequence names in the protein alignment with the names in the DNA file. Only DNA sequences with a corresponding protein in the alignment will be used as input for pal2nal.pl. This is useful, because it means that different subsets of the aligned sequences could be aligned by pal2nal.pl, using a single unaligned DNA file.

Using the MAAFT FFT-NS-i alignment as an example,  choose Edit --> Select All.

To run pal2nal.pl, choose Alignment --> pal2nal, and choose the DNA filename.

The aligned DNA sequences appear in a new blnalign window:

 
Again we see the two subfamilies of defensins, with codons aligned to the corresponding amino acids in the protein alignment. This alignment could be used directly for phylogenetic analysis.

5. Manual refinement of multiple alignments.

Alignments can be edited directly in blpalign or blnalign. By default, only gaps can be edited out, although it is possible to delete amino acids or nucleotides if protections are changed (Edit --> GetInfo).

Shifting gaps for a group of sequences in an alignment could potentially improve the alignment.

The T's highlighted in blue could just as legitimately line up with T's in the sequences above. In other words, it might be just as good an alignment if the GTT-- motif was changed to G--TT. As long as the number of gaps is conserved, the alignment could easily be revised.

We can move the block over as follows. First select all of the sequences in the block by name. Click on the topmost sequence in the block, MF289365, hold the shift key, and click on EU958634. We can make these sequences function as a group by choosing Edit --> Group. The blpalign window will now look like this:

First, insert two gap characters (--) in any sequence in group 1  to shift these amino acids to line up TT with the other occurrences of TT.  You will notice that insertion of the gaps puts the rest of the sequences downstream of the insertion out of alignment.

To restore the alignment, use the Backspace or Delete key to remove the two gaps to the right of the TTs. You can now see the amino acid motifs KV-AEA... lining up with the rest of the sequences.

6. Displaying and printing multiple 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. 

As an example, we'll print out just the sequences in group 1 from the previous section. Choose Alignment --> REFORM.

By default, the output will print a consensus sequence at top, and only show in the alignment those amino acids that differ from the consensus.

                  10        20        30        40        50        60        70
          --MAxSRR--ma--ApvlvL-l-l-lLV-ATElG--TTKV-AEARHCLSQSHRFKGlCmsSnNCANvCqT
MF289365:C....f.....v....sa.l.....v.......m.......................p.vr.g......k.
MF289364:C....f.....v....sa.l.....v.......m.......................p.vr.g......k.
MG970570:C....l.....v....sa.l.....v.......m.......................p.vr.g......k.
AP006168:C....p......v...safl....a.i......m.......................m.v.........r.
HG792392:C....p................m................................................
LN650980:C....p.............................................................a...
EU952901:C....l.................................................................
EU958628:C....l................m................................................
EU958634:C....l................m................................................

                  80        90
          -EnFPgGECkaeGatRKCFCKkiC
MF289365:C.....d...qtq.le......rv.
MF289364:C.....d...qtq.lq......rv.
MG970570:C.....d...qtq.le......rv.
AP006168:C..s..d....sh.le.......v.
HG792392:C........................
LN650980:C........................
EU952901:C........................
EU958628:C........................
EU958634:C.........r..............

 

                  10        20        30        40        50        60        70
          --MAxSRR--ma--ApvlvL-l-l-lLV-ATElG--TTKV-AEARHCLSQSHRFKGlCmsSnNCANvCqT
MF289365:C--mafsrr--va--asalll-l-lvllv-atemg--ttkv-aearhclsqshrfkgpcvrsgncanvckt
MF289364:C--mafsrr--va--asalll-l-lvllv-atemg--ttkv-aearhclsqshrfkgpcvrsgncanvckt
MG970570:C--malsrr--va--asalll-l-lvllv-atemg--ttkv-aearhclsqshrfkgpcvrsgncanvckt
AP006168:C--mapsrr--mv--asafll-l-a-ilv-atemg--ttkv-aearhclsqshrfkgmcvssnncanvcrt
HG792392:C--mapsrr--ma--apvlvl-m-l-llv-atelg--ttkv-aearhclsqshrfkglcmssnncanvcqt
LN650980:C--mapsrr--ma--apvlvl-l-l-llv-atelg--ttkv-aearhclsqshrfkglcmssnncanacqt
EU952901:C--malsrr--ma--apvlvl-l-l-llv-atelg--ttkv-aearhclsqshrfkglcmssnncanvcqt
EU958628:C--malsrr--ma--apvlvl-m-l-llv-atelg--ttkv-aearhclsqshrfkglcmssnncanvcqt
EU958634:C--malsrr--ma--apvlvl-m-l-llv-atelg--ttkv-aearhclsqshrfkglcmssnncanvcqt

                  80        90
          -EnFPgGECkaeGatRKCFCKkiC
MF289365:C-enfpdgecqtqglerkcfckrvc
MF289364:C-enfpdgecqtqglqrkcfckrvc
MG970570:C-enfpdgecqtqglerkcfckrvc
AP006168:C-esfpdgeckshglerkcfckkvc
HG792392:C-enfpggeckaegatrkcfckkic
LN650980:C-enfpggeckaegatrkcfckkic
EU952901:C-enfpggeckaegatrkcfckkic
EU958628:C-enfpggeckaegatrkcfckkic
EU958634:C-enfpggecraegatrkcfckkic


 

Jalview is a feature-rich sequence alignment analysis system. 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 Blosum62 color scheme shows conservation, as measured by Blosum62 matrix scores at each position.

The alignment can be written in paginated form, suitable for printing, by saving in a variety of formats.

Jalview can also do complete alignments from unaligned sequences. For a full description of the capabilities of Jalview, see http://www.jalview.org/help.html.