Viral bioinformatics: Multiple sequence alignment – Base-By-Base (BBB) editor

This week’s addition to the virology toolbox was written by Chris Upton (Disclaimer: BBB was developed in the Upton lab, so this is a biased review.)

This will be a multi-part posting describing the key features of BBB.

Base-By-Base (BBB) is a Java (platform independent) multiple sequence alignment (MSA) editor. Development was begun many years ago to provide a virologist-friendly tool to work with MSAs of proteins, gene sequences and also genome sequences (up to about 500 kb). Over the years we have added many unique features, as they have been needed by our own research with a variety of virus genomes; development has been driven by the users – who spend a lot of their day looking at a variety of MSAs!

BBB is available here.

The program is Open Source and freely available to all academic labs.

Help files:

Key features:

  1. The program edits MSAs and uses a unique system to display differences between sequences in a MSA. This makes it easier for the user to spot mis-aligned regions that need correcting (Figure 1A-D). The differences can be from adjacent pairs of sequences, sequences compared to a consensus, to the top sequence (Figures 1A and B).
  2. Sequences can be temporarily hidden/revealed. See “eyes” at left of window.
  3. 3-frame translations can be displayed for DNA sequences (Figure 2A).
  4. Top or Bottom strand can be shown for DNA sequences; compare Figures 2A and B.
  5. Sequence annotations can be read from a GenBank file, or added by the user (Figure 3). Users can also use a text window (Edit menu: Edit MSA notes) to jot down notes about an alignment; these are saved with the alignment in the .bbb file.
  6. You can also edit sequences! i.e. change the nucleotides or amino acids; see Figure 4. This can be very useful when you need to edit an assembled/annotated sequence for an occasional sequencing error.Figure 1A.  Differences between sequences are high-lighted. Blue=nt substitution; Green=nt deletion; Red=nt insertion.

Figure 1A. Differences between sequences are high-lighted. Blue=nt substitution; Green=nt deletion; Red=nt insertion.

Figure 1B. Edited version of Figure 1A (my opinion).

Figure 1C. Same alignment as 1B, but differences are set for compare against top sequence.

Figure 1D. Same alignment and differences setting, but Sequence-2 has been moved to the bottom of the alignment (use arrows on left edge of window).

Figure 2A. Two sequences have been hidden. 3-frame translation is shown. Top strand is shown by default.

Figure 2B. Switched to bottom strand display (use top right button; 5’Top3’ in Fig. 2A). Notice direction of arrows in aa translation.

Figure 3. Gene annotations from GenBank file (pink); mouse-over gives gene annotation (#083) and nucleotide position. User-added comments: Blue=comment on Top stand; comments associated with strand not currently displayed are shown as outlines.

Figure 4A. Editing a sequence. First, select a region.

Figure 4B. Editing a sequence. Second, delete the selection.

Figure 4C. Editing a sequence. Third, add new nucleotides.


  1. Save alignments as .bbb files on your local computer. These documents can be reloaded back into BBB.
  2. You’ll find the Preferences menu, under the File menu.
  3. You can edit the names of the sequences (Edit menu)
  4. Paper+Pencil icon is for editing; Paper+arrow icon is for selecting.

Viral Bioinformatics: Multiple sequence alignment – Jalview

This week’s addition to the virology toolbox was written by Chris Upton

The Jalview package: a multiple alignment editor.

This software is primarily aimed at the alignment of protein sequences. Some of the key features are:

  • It allows you to edit the alignment
  • It has functions to display associated protein structures
  • It can connect to software to predict protein secondary structure
  • It’s under active development
  • Jalview has great documentation and tutorials
  • More: Overview, Documentation


  1. Although you can install Jalview on your computer very easily, using the Start with Java Web Start button is even easier and ensures you always have the latest version of the software.
  2. There is also an Applet version of Jalview that is intended to be an alignment viewer – it doesn’t have all the functionality.

If you use Jalview in your work, you should cite the Jalview 2 publication:

• Waterhouse, A.M., Procter, J.B., Martin, D.M.A, Clamp, M., Barton, G.J (2009), Jalview version 2: A Multiple Sequence Alignment and Analysis Workbench. Bioinformatics 25:1189-91.

• Clamp, M., Cuff, J., Searle, S. M. and Barton, G. J. (2004), The Jalview Java Alignment Editor. Bioinformatics 20: 426-7.

Viral bioinformatics: Introduction to multiple sequence alignment

This week’s addition to the virology toolbox was written by Chris Upton

Generating multiple sequence alignments (MSA) is one of the most commonly used bioinformatics techniques. The “sequences” to be compared can be DNA (promoters, genes, genomes) or proteins. Note that the length and number of sequences to be aligned has an impact on the methods (algorithms) that can be used; what is suitable for aligning 20 proteins probably won’t work for alignment of 5 poxvirus genomes (200 kb each).

Some useful links:

So you see, there lots of options (did you say: “too many!”?). Further confusion may arise because 1) the same algorithm may be used in many different software programs, and 2) referencing a software package may give no clue to the algorithm used. For many molecular biologists, Clustal is synonymous with sequence alignment. However, newer algorithms such as T-Coffee and MUSCLE are often offered in current software packages, and may be faster and more accurate.

Specialized alignment tools are almost always needed for long, genome sized DNA sequences.

In this set of posts, I’ll provide some information on favorite general MSA tools (that are free) that should be useful to the average molecular virologist. The lists noted above provide a multitude of tools, but many are for specific analyses.