C O V A R I A T I O N A Hypercard stack for phylogenetic comparative analysis of aligned RNA sequences. THE "COVARIATION" PROGRAM Covariation is a Macintosh Hypercard program which can be used to identify compensating base changes or covariations within aligned RNA sequences. Covariation reads the sequence alignment, compares the patterns of nucleotides a each position within the alignment, and displays the relationships between the patterns graphically (the required format of the alignment, and the details of the output, will be described later). If the alignment contains enough sequences with enough variation in sequence, helical regions are shown as diagonal stripes, much like those produced by "diagon"-type matrix analysis of sequence similarity. Although Covariation is a Hypercard "stack", it runs more like an application: the standard Hypercard menus are replaced by menus for Covariation, and the normal Hypercard commands are not available (or required). Covariation appears as a single dialog box (actually a card, of course), rather than a stack of cards. The user need not be familiar with Hypercard, therefore, to use Covariation. SYSTEM REQUIREMENTS Covariation is written in HyperTalk, and requires Hypercard version 2.0, which in turn requires at least a Macintosh plus, 2Mbytes of RAM, and System v6.0.5. Covariation is also compatable with Hypercard v2.1 and System 7.0, and includes Balloon Help for System 7 users. A hard disk is highly recommended (i.e. required) because of the large size of the System, Hypercard, and the output files generated by the program. Although Covariation will run on any Macintosh capable of running Hypercard 2.0, it's best to run it on as fast a Macintosh as possible. On slower Macs, such as the Plus, SE, portable, or Classic, be prepared to wait hours, even days, for a single analysis to run to completion. It's also nice to have a large screen for manipulating sequence alignments and the output files generated by Covariation. INSTALLING & RUNNING COVARIATION In order to use Covariation, just copy the Covariation folder from this floppy disk to your hard disk - put it anywhere you want. Make sure that Hypercard v2.0 (or greater) and Home are also on the hard disk (or at least available, i.e. on a file server). Just double-click on the Covariation icon, & you're off and running! To install Covariation as an icon in your Home stack (this is not a requirement, but is a convenience if you use Hypercard alot), create a new button in your Home stack, and select an icon which reminds you of Covariation (or edit one). Rename the button "Covariation", and link this button to the Covariation stack. (If this makes no sense to you, don't bother with it. The manuals that come with Hypercard describe the method - look it up there.) WHERE TO GET COVARIATION OR UPDATES Covariation is available on internet by anonymous FTP from bio.indiana.edu, by E-mail from brownjw@iubacs.bitnet or jwbrown@bio.indiana,edu, or by regular mail from the author (enclose a single 800K formatted Macintosh floppy disk). Files obtained electronically will be stuffed and binhexed, and so require translation using Stuffit. For further information contact the author at: Dr. James W. Brown, Department of Biology, Indiana University, Bloomington, IN 47405 USA, or at the BitNet E-mail address given above. Please feel free to copy or give Covariation away to anyone who might want it, but at NO CHARGE WHATSOEVER! Please keep all of the accessory files with the program, & only give any unaltered copies of the program. This program is NOT public domain - all rights to the program are retained by the author. WHAT COMES WITH COVARIATION The following files should have come in the Covariation folder: Covariation v2.6 - the analysis program itself, a Hypercard stack. Covariation Printer - a utility to print out large result files, a Hypercard stack. READ ME! - this documentation file. Sample alignment - an example alignment input file. compensatory (list) - an example output file. compensatory (matrix) - another example output file. covariation (list) - yet another example output file. covariation (matrix) - still yet another example output file. If these files did not come with your copy of Covariation, contact the author for a complete copy of the most up to date version of Covariation. OTHER USEFUL PROGRAMS Covariation requires that you provide it with a multiple-sequence GenBank format file for input. Many alignment editors can save the alignment in GenBank format, e.g. AE2, GDE, and SeqApp. SeqApp, a Macintosh-based multiple sequence editor (alignment editor), is available from Don Gilbert (same address as the author) or from ftp.bio.indiana.edu. Although this program is unfinished (alpha release), it works pretty well and is a great way to generate alignments for use with this and many other programs because, amoung other things, it uses GenBank format. I highly recommend this alignment editor. Users of the UWGCG package can also generate multiple-sequence GenBank format files using the appropriate command. DNA Translator and Aligner are two Hypercard stacks that are very useful in generating sequence alignments and transferring files to and from various file formats. These stacks are available from J. Ernisse (usergdef@um.cc.umich.edu or usergdef@umichum.bitnet), Museum of Zoology, Univ. of Michigan, Ann Arbor, MI 48109 (see CABIOS 8:177-184 for descriptions of the stacks). BBEdit is a basic, "Bare-Bones" editor that is very useful in editing & printing files with long lines, such as the output files from Covariation. BBEdit is shareware, & is available from Rich Siegel or various ftp sites. DISCLAIMER DISCLAIMER DISCLAIMER DISCLAIMER DISCLAIMER DISCLAIMER DISCLAIMER Every effort has been made to ensure that Covariation will function as described, but the author makes NO GUARANTEE as to the suitability of Covariation for any particular use, and is distributed AS IS. Any results from the use of Covariation are the responsibility of the user. The author does not take responsibility for any crashes, mishaps, weird phenomenon, or any other outcome of the use of this program.