To all: Thanks for the many replies to my request for advice on sequence alignment software. Wow. There seems to be some agreement around BioEdit, T-Coffee, and a few others, but I think it easiest if just include a compiled list of the replies, edited for redundancy, for the most part. --Bob ---- Hi Robert, I have not used it extensively, but T-Coffee has worked nicely the times I have tried it.  Both webbased http://www.tcoffee.org/ and possible to download http://www.tcoffee.org/Projects_home_page/t_coffee_home_page.html. Thomas Källman (PhD student) Program in Evolutionary Functional genomics Evolutionary Biology Centre Uppsala University Norbyvägen 18D, 752 36 UPPSALA SWEDEN ---- Try MEGA3 at www.megasoftware.net. There is also a paper describing the software at that site. Sudhir --------- Sudhir Kumar (s.kumar@asu.edu) Director, Center for Evol Func Genomics Biodesign Institute. BDA 240 Arizona State Univ Tempe, AZ 85287-5301 www.kumarlab.net ---- There are quite a few free packages available.  I like multialign (http://bioinfo.genopole-toulouse.prd.fr/multalin/multalin.html).   It is web-based and allows you to view your sequences in a variety of formats.   Joe Felsenstein manages a great website that contains a list of available software http://evolution.genetics.washington.edu/phylip/software.html#Management  ---- MAFFT, MUSCLE, and MAVID are three of the latest programs widely used for alignment. There are quite a few webservers so that you do not have to align locally on your machine. For links, see my dedicated website at http://research.amnh.org/users/koloko/softlinks (click "alignment"). Best, Sergios ---- Sergios-Orestis Kolokotronis Institute for Comparative Genomics American Museum of Natural History Central Park West @ 79th St. New York, NY 10024 -USA- tel +1 212 313 7648 koloko@amnh.org http://koloko.net Dept. of Ecology, Evolution, and Environmental Biology (E3B) Columbia University sk2059@columbia.edu   ---- Dear Robert, If the alignment is difficult (many indels), we start with Clustal, which you can use on the web or download free. We finish the alignment and check it in MacClade (not free but worth the price), where we use the nice color view to check and fine-tune the alignment by hand. MacClade will also translate the sequences, and for protein-coding genes it is best to align the amino acid sequences first. Indels due to sequencing artefacts are quickly spotted as frameshifts. I'l guessing that there is freeware that you can use to translate, color, and align sites, although it presumbly won't do all the other things that MacClade does. For rDNA it is best to take secondary structure into account when doing the alignment, but that is a whole different problem which others can tell you more about. We are only just getting started on it. Good luck! Bill C. William Birky, Jr. Professor of Ecology and Evolutionary Biology Member, Graduate Interdisciplinary Program in Genetics Department of Ecology and Evolutionary Biology Biological Sciences West The University of Arizona Tucson, AZ office: 520-626-6513 lab: 520-626-5108 http://eebweb.arizona.edu/faculty/birky/home.html ---- hi Bob- CLUSTALW, TCOFFEE, MUSCLE, KALIGN, PROBCONS, MAFFT they all have web interfaces if you google for them. http://www.ebi.ac.uk/muscle/ http://www.ebi.ac.uk/clustalw/ http://www.ebi.ac.uk/t-coffee/ http://www.ebi.ac.uk/kalign/ http://www.ebi.ac.uk/mafft/ http://bioperl.org/wiki/Multiple_sequence_alignment http://en.wikipedia.org/wiki/Multiple Jason Stajich Miller Research Fellow University of California, Berkeley lab: 510.642.8441 http://pmb.berkeley.edu/~taylor/people/js.html http://fungalgenomes.org/ ---- There is a large number of free multiple sequence alignment tools: - clustalw - well know often used, but gives not always good results (online: http://www.ebi.ac.uk/clustalw/) - t-coffee - combines local and global alignment techniques and, hence, tries to circumvent mistakes that may happen in clustalw. (http://www.tcoffee.org/Projects_home_page/t_coffee_home_page.html, contains also links to webservers, e.g. http://www.tcoffee.org/) - m-coffee - new approach combining t-coffee with other multiple sequence alignment methods. (available online at http://www.tcoffee.org/ as well, the paper http://nar.oxfordjournals.org/cgi/content/abstract/34/6/1692 also contains links to almost all multiple sequence alignment methods I know. And it includes some comparison.) Some new and fast ones seem to be: - MUSCLE: multiple sequence alignment with high accuracy and high throughput http://nar.oxfordjournals.org/cgi/content/abstract/32/5/1792 and - MAFFT version 5: improvement in accuracy of multiple sequence alignment http://nar.oxfordjournals.org/cgi/content/abstract/33/2/511 improved version of: MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform http://nar.oxfordjournals.org/cgi/content/abstract/30/14/3059 Several programs are available online at the EBI via: http://www.ebi.ac.uk/Tools/sequence.html However, no matter how well those programs claim to be, you always have to check the resulting alignments. The resulting alignments usually contain errors which sneak in because the methods try to heuristically optimize an objective function and cannot know about all biological constraints like, e.g., the relevance of certains Cysteins in certain proteins. Here we have to help the algorithms with biological knowledge and correct the alignment... which on distantly related sequences might need quite some time and sometime expertise. We have to be aware that in subsequent phylogenetic and other other analysis "garbage in" usually also results in "gabage out". Cheers, Heiko Heiko Schmidt Center for Integrative Bioinformatics Vienna (CIBIV) Max F. Perutz Laboratories (MFPL) Dr. Bohr Gasse 9 Tel.: ++43 +1 / 79044 - 4586 A-1030 Vienna, Austria Fax.: ++43 +1 / 79044 - 4551 Email: heiko.schmidt(AT)univie.ac.at http://www.cibiv.at/   ---- You can have a look at our Free Workbench http://www.clcbio.com/index.php?id=3D487 It provides multiple alignment using the following algorithms: * Two proprietary algorithms * ClustalW * Muscle * T-Coffee * MAFFT * Kalign Cheers -- Roald Forsberg Senior Scientific Officer CLC bio A/S Gustav Wieds Vej 10 8000 Aarhus C Denmark http://www.clcbio.com http://www.clcbioconsulting.com/roald.html rforsberg@clcbio.com Contact numbers: Telephone: +45 70 22 32 44 Mobile: +45 61 65 63 20 Fax: +45 70 22 55 19 Robert.Marra@po.state.ct.us