Both will work with sequence alignments in many forms, including GenBank and the cross-platform FASTA (FAST Alignment) format. In these examples, we will use the FASTA format. Here is an example of that format:

>gi|112927|sp|P05067|A4_HUMAN Amyloid beta A4 protein precursor (APP) (ABPP)
MLPGLALLLLAAWTARALEVPTDGNAGLLAEPQIAMFCGRLNMHMNVQNGKWDSDPSGTKTCIDTKEGIL
QYCQEVYPELQITNVVEANQPVTIQNWCKRGRKQCKTHPHFVIPYRCLVGEFVSDALLVPDKCKFLHQER
MDVCETHLHWHTVAKETCSEKSTNLHDYGMLLPCGIDKFRGVEFVCCPLAEESDNVDSADAEEDDSDVWW

The above is a portion of the sequence of a protein. The first line must always begin with > and is a comment or identification line. This is followed by the sequence, using the one letter codes for bases or amino acids. The length of the lines is irrelevant.

BioEdit by itself is capable of pairwise alignment only. However, the program ClustalW comes with it, is installed in a subdirectory, and can be run from within BioEdit to create multiple alignments. Consult the Help file to learn how to configure this option. JalView does multiple alignments by accessing the ClustalW web site directly.

To do a pairwise alignment in BioEdit:

• Use File/Open to read in the first sequence, choosing the appropriate file format

• Read in the second with File/Import

• Both sequences will now appear in the main window

• If you are aligning nucleic acid sequence, the program will automatically chose an Identity matrix for scoring; for proteins, you must choose your scoring matrix (explained below) using Sequence/Similarity Matrix. Blosum62 is the default.

• Hold down the Control key and click on the titles of both sequences to select them

• Now select Sequence/Pairwise Alignment. You will be offered two options
  • Optimal global alignment; this will maximize the number of matches, allowing gaps

  • Allow ends to slide keeps the sequences intact and slides one sequence as a whole relative to the other

• Make your choice; a window will pop up with the score for your alignment and the aligned sequences will appear in the main window:

To make a nicely colored version of the alignment, select File/Graphic View

• You can now fill in background colors for identical and similar residues
  • Click on the boxes labeled "Similar" and "Identical" to choose background and foreground colors

  • Check Identity/similarity shading (lower left) to have your choices take effect

• Other formatting choices:
  • Position the titles left or right or truncate them using the appropriate check boxes

  • Use the check box to group the residues in blocks of ten, and the scroll box to limit the width of the display to your window

  • Choose File/Copy as Bitmap or File/Copy as Enhanced Windows Metafile to copy your prettied up alignment to the clipboard. From there, you can paste it into Word or your favorite graphic program.

Here's what the Graphic View window will look like when you're done:

BioEdit will also translate DNA or RNA to protein, find cDNA or the reverse complement, do a nice analysis with bar graph of the percentages of the four bases, and otherwise characterize your sequence.

For example, with your human elastase gene loaded and selected, choose Sequence/Nucleic Acid/Translate/Frame 1. You will get a new window containing the translation. Try this when you have a sequence displayed in Graphic View also.

To do an alignment in Jalview, choose File/Input Alignment/from File. The result will look like this:

In the new window that has appeared, choose File/Add Sequences/from File.

Select both sequences by holding down Ctrl and clicking on their titles. Then choose Calculate/Pairwise Alignments. The result looks like this:

Click the "View in alignment editor button at the bottom of this window. You can now choose to color your alignment by any of a variety of schemes. I chose the Clustalx scheme, which assigns each amino acid a color. I then chose Wrap from the Format menu to produce:

You can save your alignment as a picture by choosing Export Image from the file menu; this gives you the choise of a web page (html), a Postscript file (eps) or a Word-compatible picture png).

Multiple sequence alignments are done by reading in all of the files of interest and choosing Web Service; select your preferred server from the menu.

Here are the URLs for a number of web servers that will carry out alignments on sequences that you submit:

Easiest is to have your multiple sequences combined into a single file: simply use NotePad or any text editor. Paste one FASTA file after another into a single FASTA file (include the title lines), and use the upload feature. Alternatively, you can just copy and paste, one after the other, into the alignment window.

Allignments produced by any of these servers can be read into BioEdit or JalView and formatted to suit.

Similarity Matrices Any alignment requires some method of judging the quality of fit between the two or more sequences. Any scoring scheme must account for gaps (which we saw in our alignment of the two elastase genes), substitutions, and insertions and deletions.

For nucleic acids, the scoring scheme can be relatively simple; since only for characters occur in the sequences, substitutions are generally not present. BioEdit uses the following values:

JalView penalizes -12 for opening a gap and -2 for a gap extension.

Such a scoring system allows us to compare several possible alignment and choose the "best" - the one with the highest score.

In proteins, amino acid substitutions occur frequently, especially among amino acids of similar physicochemical properties. Alanine (methyl side chain) and valine (isopropyl side chain) are common substitutions for each other, that can occur without making a significant difference in the properties of a protein. Hence, more complicated schemes than that above are required to score alignments.

Perhaps the most widely used scoring matrices for proteins are the BLOSUM (blocks substitution matrix) developed by S. and J. G. Henikoff ["Amino acid substitution matrices from protein blocks", Proc. Natl. Acad. Sci. 1992, 89 10915-10919.] .

• From regions of closely related proteins that can be aligned without gaps, they calculated the ratio of observed pairs at any position to the number expected from overall amino acid frequencies

• The results are expressed as the log of this ratio, multiplied by 10 to avoid decimals

• Depending on the particular sequences used, several matrices have been developed; BLOSUM62 is the most common.

A few explanatory comments:

• The X represents an unknown amino acid, missed in the sequencing

• The asterisk (*) is a gap initiation penalty

• Glutamic acid and aspartic acid occur as both the acid and the amide (glutamine and asparagine, respectively). Hence aspartic acid is both B and D, and glutamic acid is E, Q, and Z.

To look up the value corresponding to the substitution "N->W" (an Asparagine is replaced by a Tryptophan), go to the line of the table labeled "N" and follow this line until you get to the column labeled "W": -4. The substitution "N->W" is not very likely; that is, it does not occur often in closely related proteins. On the other hand, the substitution "V->I" (BLOSUM score: 3) is very likely.

BioEdit itself can only do pairwise alignments. However, the download includes a copy of the multiple alignment tool ClustalW, which can align many sequences. You can use this directly, or configure BioEdit to run it for you.