May 3, 1999
To: Darcy Kelley
Fm: Joe Thornton
Re: Sequence analysis software

Dear Darcy:

In response to your request for information on sequence analysis software, here is a brief note on what I use, its strengths and weaknesses, and a recommendation for some programs I feel the department should obtain. This is not intended as an exhaustive evaluation; although I do quite a bit of analysis of DNA and protein sequences, this list is circumscribed by my personal experience.

• GCG -- for miscellaneous applications. I use GCG to a very limited extent, because I find it user-unfriendly. While it contains many individual applications, I’ve yet to encounter a need that I can’t fulfill with one of the following easier-to-use programs. Interestingly, while GCG has been a standard tool for many for molecular biologists, virtually no one in the evolutionary biology community (including those who work primarily with sequences) uses it.

• CLUSTAL-X -- Multiple sequence alignment. Clustal includes a powerful and reliable algorithm for alignment of protein and nucleotide sequences. It is easy to use, and it is configured with a generally effective set of default alignment parameters. It is easy to customize the parameters, which is important to anyone who doesn’t want alignment to be a "black boxed" process. The Macintosh version (and I believe the PC version, as well) allow the aligned sequences to be viewed on screen within the program, which makes it much more convenient than GCG; it also exports its alignments in a variety of text-based formats, including GCG, NBRF/PIR, Phylip, GDE, and Clustal. Clustal is free from EMBO’s web-site, and it is available for Mac, PC, and Unix.

• MEGALIGN -- sequence alignment. Megalign is part of Lasergene’s commercial DNA-Star package. It includes the Clustal algorithm, along with TreeAlign/Jotun Hein, another reliable alignment method that refines its guide trees using a parsimony criterion -- a distinct advantage philosophically over Clustal’s neighbor-joining approach. Megalign also allows alignments to be edited/adjusted on-screen, and alignments can be printed out with color-coding, a consensus, and sequence numbers. It also allows one-click translation/reverse translation between nucleotide and amino acid sequences -- a very useful feature for maintaining reading frame during the alignment of coding sequences.

• PAUP 4.0 -- Phylogenetic analysis. The new PAUP does everything one could ever want in a phylogenetic analysis program. It performs both parsimony and maximum likelihood analyses, with full control over parameters and more options than any one person will ever use. It also performs distance-based tree reconstructions, including UPGMA and neighbor-joining methods, for those looking for a quick-and-dirty (but unreliable) tree. It does bootstraps, distance matrices, character-change matrices, and everything else you could ever think of. It is an easy to use menu-driven program in its Mac incarnation. It is also available for Unix and for windows. I assume the latter is menu-driven, though I have never used. The program is commercially available from Sinauer, and it is cheap (about $100, I think). For those who want to do more extensive evolutionary character analysis, the software of choice is MacClade (also from Sinauer), but I doubt anyone in this department will undertake this kind of work. Many people also use Phylip (University of Washington) for a few features not found in PAUP; overall, it is less powerful and complete than the new PAUP.

• BLAST searches -- I do this on-line through NCBI.

• GENBANK searches -- I do this on-line through NCBI.

• EDITSEQ -- basic sequence editing. I find EditSeq, part of the DNAStar package from Lasergene, extremely useful for basic sequence manipulation. It locates reading frames, translates, reverse translates, reverse-complements, and saves files in various formats. It also gives statistics on the sequence in the file -- base or amino acid composition, melting temperature, etc.

• SEQUENCHER -- Analysis of automated sequence files. This is the most important program that the department is currently lacking. It is the best available software for dealing with sequence files provided by an automated sequence machine or analysis center. It offers a very powerful and easy-to-use method for automatically aligning sequence fragments (including those read on opposite strands) and assembling and editing of contigs directly from automated sequence output files. It allows easy location of ambiguous sites in a sequence, and a single click allows the user to view the electropherograms at that base in order to judge which sequence is most reliable. It also finds ORFs, translates in multiple frames, analyzes restriction sites, locates common vector sequences, screens for transposon sequences, trims dirty ends from sequence files based on ambiguity in the pherogram, performs heterozygote analysis for mutation studies. No one who does or has automated sequencing done for them should be without this program.