How to use multiple sequence aligners in GBench

For multiple alignment creation Genome Workbench Tool includes four different third party alignment programs:

• CLUSTAL (omega and classical): http://www.clustal.org/
• Kalign: http://msa.sbc.su.se/cgi-bin/msa.cgi We found that kalign version 2.04 that we used does not compile on Windows. We had to make minor technical modifications to port it. NCBI modified source code and binaries are available at https://ftp.ncbi.nlm.nih.gov/toolbox/gbench/kalign.
• MUSCLE: http://drive5.com/muscle/
• MAFFT: http://mafft.cbrc.jp/alignment/software/

Multiple sequence alignment creation

1. Create set of DNA or protein sequences in fasta format Example fasta files:
2. DNA: 16SRNA_Deino.fa (88 sequences)

3. Proteins: Cu_Zn_SOD.fa (60 sequences)

4. Upload fasta file into a new project via File > Open > File Import, select FASTA sequence files for file format, select path to the fasta file > Next > Use default settings > Next > Create a new Project_(add checkmark to "Create a Folder for the items" checkbox and specify subfolder name)> Finish.

1. Select all objects (Shf+click) in newly created subfolder or Data folder in the Project view and open Tools menu (use double click, or Tools icon, or Right Click/Run Tool option).

1. In Run Tool dialog select preferable multiple sequence aligner and click next.

1. Run Tool dialog for selected aligner opens with all sequences selected (if sequences are not selected use Select All button).

2. Select local path to the aligner executable. For the external tools such as aligners you need to download and install the tools from their corresponding sites. For many tools binary executables are available for all popular platforms.

1. Click Finish and wait for alignment creation. Since alignment algorithm runs as an external process, command prompt window will pop up with the tool running.

1. Created alignment will be automatically added to the Tool Results subfolder in the current project.

After multiple alignment has been created, it can be opened in the Multiple alignment view (see below) or/and used for tree construction, see How to create phylogenetic tree in GBench.

Open Alignment in Multiple Alignment view

Double click on alignment in Project View (or select it by right click, it will open Right click menu). In the menu select Open New View, in Open View dialog select Multiple Alignment View, and click Next to open alignment.

The image below demonstrates protein alignment created by MUSCLE.

Coloring is disabled by default. To select coloring method perform right click anywhere in the alignment, it will open menu, select Coloring > Select Method

In the Alignment Scoring Methods dialog select preferable method.

The image below shows protein alignment created by MUSCLE and colored using Rasmol amino acid colors.

The image below shows DNA alignment created by Kalign and colored by Frequency-Based Difference method.

Examples for all aligners can be seen in the saved projects:

• project_60_protein_seq.gbp
• project_88_dna_seq.gbp

Export of alignments to FASTA alignment file

You can export the alignment in FASTA format.

1. Select the alignment object in your project (Project View)
2. Use File->Export menu or context menu Export
3. Use Export Dialog to export as FASTA Alignment file and specify the filename

See also

Multiple Sequence Aligners in Genome Workbench video tutorial

Coloring methods in Multiple Alignment View tutorial

Coloring legend