Sequence alignment

Powerpoint slides are here

Pairwise alignment

A) DOT PLOT See the Dotlet exercises form last week.

The easiest way to align two sequences is to use a dotplot. In its most straight forward implementation the two sequences to be aligned are written along the coordinate axis.

In more realistic implementations a window of 5 to 20 nucleotides or amino acids is slid along one of the axes (i.e., sequences) and compared to every possible window on the other axis (sequence). The dot intensity is adjusted to reflect the percent identity (or similarity) in the two windows.

Optimal global and local alignments.

There are many different algorithms to calculate pairwise sequence alignments. For two sequences it is "easy" to calculate an optimal global alignment. (According to the motto: "It can be easily shown" -- see here, and here -- the links refer to a bioinformatics course given at the Univ. of Munich). The so called Needleman-Wunsch algorithm is widely used, it optimizes a positive alignment score, a related (and under some conditions equivalent approach) is to minimize the differences between to sequences.

 

Multiple Sequence Alignments

CLUSTAL, CLUSTALW and CLUSTALX

Usually global alignments are the easiest to calculate (local see below)

One of the easiest to use, most sophisticated, and most versatile alignment programs is clustalw

(Higgins DG, Sharp PM (1988) CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 73:237-244;
Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22, 4673-4680).

Clustalw runs on all possible platforms (unix, mac, pc), and it is part of most multiprogram packages, and it is also available via different web interfaces (for examples here, here, and here). 

Clustalw uses a very simple menu driven command-line interface, and you also can run it from the command line only (i.e. it is easy to incorporate into scripts for repeated analyses.)

Clustalx uses the same algorithms as clustalw.  However, it has a much nicer interface, it displays information on the level of similarity, and it uses color in the alignment.  Especially for amino acids the use of color greatly enhances the ability to recognize conservative replacements. Clustalx is available for different platforms at the ebi's ftp site (follow your platform, clustalx is stored in the clustalw folders)

Clustal reads and writes most formats used by different programs.  The easiest format is the FASTA format:

> name of sequence or any other information goes in the first line. This line starts with ">". The line can be longer than 80 characters. The first line ends with the first paragraph sign.p
The second line contains the sequence itself; numbers and other non standard characters are ignored. Be careful if you download sequences. Often the transfer programs introduce paragraph signs every 100 characters, and the end of a command line frequently ends up as the beginning of the sequence.
All sequences to be read should be in a single file.

(sample clustalw input file)

(sample clustalw output file)

Clustal also reads aligned sequences.  If you input aligned sequences you can go directly to the tree section.
!! Be careful if you make a mistake, and the sequences are not aligned, your tree will look strange!!
!!! ALWAYS CHECK YOUR ALIGNMENT!!!

Clustal also is useful to reformat and edit alignments, it is very forgiving in reading formats, e.g., you can open the clustal format (*.aln) in a text editor and delete columns and reload the file into clustalw, and output it in the other formats available.

For calculating an alignment, you can select different substitution matrices, and gap penalties (end-gaps can be considered differently!)

Clustal is better than its reputation. It is doing a great job in handling gaps, especially terminal gaps, and it makes good use of different substitution matrices.

To align sequences clustal performs the following steps:

1) Pairwise distance calculation
2) Clustering analysis of the sequences
3) Iterated alignment of two most similar sequences or groups of sequences.

It is important to realize that the second step is the most important. The relationships found here will create a serious bias in the final alignment. The better your guide tree, the better your final alignment. You can load a guide tree into clustal. This tree will then be used instead of the neighbor joining tree calculated by clustalw as a default. (The guide tree needs to be in normal parenthesis notation WITH branch lengths).

NOTE that clustalw and other multiple sequence alignment programs do NOT necessarily find an alignment that is optimal by any given criterion. Even if an alignment is optimal (like in the Needleman Wusch algorithm), it usually is not UNIQUE. It often is a good idea to take different extreme pathways through the alignment matrix, or to use a program like tcoffee that uses many different alignment programs to build a consensus .

A recently very popular alternative to the clustalw is MUSCLE, especially if you have very large datasets, or if you have very divergent sequences. Like clustalw it is run from the commandline, it runs on all possible platforms, and it can generate profile alignments, i.e., if you want to align two homologous protein families, you first align them each seperately, then you take both of them and align them with one another, or you could use this to add an additional sequence to an already existing alignment. The MUSCLE homepage is here.

Below is a session with muscle in which first single alignments are created for a sequences of the V-ATPase and F-ATPase catalytic subunits seperately, then the resulting alignments are aligne with one another (also, at each step the alignments are "refined"):

muscle -in VatpA.fa -out VatpA.afa
muscle -in VatpA.afa -out VatpA.rafa -refine
muscle -in beta.fa -out beta.afa
muscle -in beta.afa -out beta.rafa -refine
muscle -profile -in1 beta.rafa -in2 VatpA.rafa -out Abeta.afa
muscle -refine -in Abeta.afa -out Abeta.rafa

The file utilized are here and here, the result is here

Other programs often used for global multiple sequence alignments
(We will not use these program in this course; if you are already confused by the information provided, skip this section):

A program available via the www is SAM (sequence alignment and modeling system) by Richard Hughey, Anders Krogh, Christian Barrett, & Leslie Grate at UCSC. The input consists of a multiple sequence file (aligned or not aligned) in FASTA format. The program uses secondary structure predictions, neighboring sites, etc. to place gaps. The program can be accessed using netscape at " http://www.cse.ucsc.edu/research/compbio/sam.html ".

If your sequences are not very similar, and if you are not able to generate a trustworthy multiple sequence alignment, you can calculate distance trees based on pairwise alignments only. The best program for this purpose is statalign from Jeff Thorne (Thorne JL, Kishino H (1992) Freeing phylogenies from artifacts of alignment. Mol Bio Evol 9:1148-1162). It runs under standard UNIX.  It's only worth your effort if you are getting gray hairs because of a dataset you cannot reliably align.

PILEUP in the GCG package generates alignments that are very similar to clustalw. The TREE programs in GCG are currently considered by many to be worthless (UPGMA).  It is planned -since over six years- to incorporate PAUP into GCG in the "near future".

TCOFFEE extracts reliably aligned positions from several multiple or pairwise sequence alignments. It requires more thought and attention from the user than clustalw, but it helps to focus further analyses on those sites that are reliably aligned. A description is here, a web interface is here (note the book advertisement at the bottom of the page).

• Advantage: you get to know your sequences and the motives that are conserved are reliably aligned
• Problems: time consuming, Macaw has the tendency to crash.

Alignments by Eye:
On PCs there is a DOS program called the Eyeball Sequence Editor (ESEE) that allows to simultaneously align nucleotide and encoded protein sequences. Needs some getting used to.

One useful sequence editor is seaview, the companion sequence editor to phylo_win. It runs on PC and most unix flavors, and is the easiest way to get alignments into phylo_win.