Greetings: Thank you to everyone who took the time to reply to my recent posting asking for programs or methods for concatenating multiple sequence alignments into one overall alignment. Several individuals asked that I share the results of my query regarding concatenating multiple sequence alignments (MSAs). First and foremost, many reminded me to make sure that the sequences in each MSA were in the same order (i.e., if MSA one contained sequences from Human, Chimp, Mouse, and Chicken in that order, the sequence in all other MSAs had to be in the order Human, Chimp, Mouse, Chicken). Second, for several of the most efficient methods to work, the sequences in the different MSAs need to have the same sequence ID as most of the programs use this information to match and stitch together the sequences. The most common and generally simplest recommendation was to append each MSA one below the other in one file and then input this into PHYLIP or PAUP as interleaved sequence. For PAUP (and MacClade and Mesquite and MrBayes) this will require that the file be a nexus-format file. For PHYLIP the file will need to be in the standard PHYLIP format. With this method the program will concatenate the sequences in the process of reading them into the analytical pipeline. PAUP and MacClade (and possibly Mesquite?) can then export the processed input file as sequential data and in several formats. Another sequence analysis program recommended by several responses was DAMBE, which has a concatenation function among its features. DAMBE can be downloaded from http://dambe.bio.uottawa.ca/software.asp. DAMBE is a Windows program, but presumably it can work in an emulator (Virtual PC) or virtual machine (Parallels desktop). Still more individuals recommended using a Perl script and/or Perl and BioPerl modules to perform the concatenation. If one is running Mac OS X then perl is an available option. Another interesting alternative was to use Excel. One can open the alignment file as text so that the sequences are in individual cells. Though not absolutely required the individual MSAs can then be copied and pasted into one spreadsheet. Then the function concatenate() can be used to concatenate the cells with the appropriate sequences. While itıs not necessary to have the sequences in the MSAs in the same order with this method it would be much easier to implement if they were. Other programs mentioned that can perform concatenation were ProSeq v 3.0 (latest development version can be downloaded from http://helios.bto.ed.ac.uk/evolgen/filatov/proseq.html), the bioinformatics workbenches CLC Gene, Protein, and Combined (fully functional demos available from http://www.clbio.com), Genious (7-day trial available from http://www.genious.com), the Se-Al alignment editor ( available from http://evolve.zoo.ox.ac.uk/software.html?id=seal), and the BioEdit Windows biological sequence editor (available from http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Cheers, Kurt Wollenberg, Ph.D. Phylogenetics and Sequence Analysis Consultant Biocomputing Research Consulting Section Bioinformatics and Scientific IT Program (BSIP) NIH/NIAID/OTIS Contractor, MSD Inc. wollenbergk@niaid.nih.gov