The entry in the mRNA textbox can be a single FASTA sequence, a single gi or accession, a list of FASTA sequences, or a list of gis or accessions. When multiple mRNAs are aligned to a single genomic sequence, the results page will start with a list of the mRNAs that were run so that you can easily jump down to see the results. You cannot mix FASTA sequences with gis or accessions.

When you have multiple mRNAs, you can elect to produce a multiple alignment of those mRNAs against the genomic sequence. If the mRNAs overlap in any region, the multiple alignment will appear at the bottom of the results page. If the mRNAs do not overlap, you will see an error message.

If you try to align sequences from two different species (or finished mRNAs to draft sequence) using the standard parameters, usually you will get many, many short exons. To force the alignments to be longer and to have more gaps and mismatches (as you will see in interspecies comparisons), the BLAST parameters need to be changed. The mismatch penalty is lowered, and the gap opening and extension penalties are lowered. This encourages BLAST to merge nearby alignments into a single exon; we find that with these parameters, the gene models look much more realistic.

Spidey tries to keep its models compact, so it has a maximum allowed intron size (two, actually -- a smaller size for internal introns, and a larger size for the first and last introns). In some cases, though, the intron size of the correct model is larger than Spidey's maximum intron size, so Spidey cannot arrive at the correct model. If you know that the introns are especially large (longer than 35kb for internal introns, 100kb for terminal introns), then you may want to try the large intron mode.

Since the genomic sequence is the one that has the splice sites, Spidey needs to know what organism the genomic sequence is from so that it can use the correct splice site matrices. Spidey has splice site matrices for vertebrates, Drosophila, C. elegans, and plants.

The minimum percent identity and mRNA length coverage are cutoffs that Spidey uses to evaluate the final alignments. If the percent identity of the final alignment is lower than the specified cutoff, or if the percent of the mRNA's length that is covered by the alignment is lower than the percent length chosen, the final alignment will not be reported.

Spidey is specialized for doing spliced alignments. After an initial BLAST search, Spidey sorts through the hits to find a set of alignments that are consistent and that cover the mRNA pretty well. Then, Spidey tries its hardest to align any pieces of the mRNA that aren't yet aligned -- to do this, it uses low-stringency BLAST as well as another type of local alignment program. Finally, Spidey uses splice matrices to adjust the boundaries of the exon alignments so that they do not overlap or underlap and so that they are adjacent to good splice sites.

Short answer: no. Longer answer: The strength of the evidence for intron and exon features annotated in Genbank varies widely. Since many of these have been found by programs, not through experimental work, it doesn't make sense to use these annotations because then Spidey would just pick up all the quirks of other alignment programs. Also, since Spidey was designed to take FASTA sequences as input, it has to be able to align sequences blindly anyway.

Everyone wants to know this one . . . I have to admit that the choice was almost an accident. I had written some functions and needed to save the file, so I had to call it something. I was listening to Moxy Fruvous' song 'Spiderman' on my headphones, and 'Spidey' just seemed right somehow, so I figured 'Spidey' would be a good temporary name until I could think up something catchier. After a while I liked 'Spidey' too much to give it up. I like to think of the program as a spider, picking its way around a web of sticky and non-sticky strands and being agile and smart enough not to get stuck (most of the time, anyway). The spider logo was done by my husband, Brian Greenlee, who is a graphic designer. I suppose the spider is also a tribute to one of my favorite musicians, John Entwistle, bassist for The Who and author of '5:15', 'My Wife', and, of course, 'Boris the Spider'.

Thanks to Bhanu Rajput and Kim Pruitt for their terrific comments and suggestions and for their insightful questions.