Nucleic Acids Research, 2005, Vol. 33, Web Server issue W507 sequence features. Similar programs for morpholino-speciﬁc design are not currently available, although the sole com- mercial supplier of morpholinos, Gene Tools, offers a free, proprietary design service (http://www.gene-tools.com/) that requires prior knowledge of the translational start codon in the mRNA and provides very limited sequence design and analysis options to the user. AMOD implements morpholino design guidelines similar to those recommended by Gene Tools, such as avoidance of nucleotide motifs that form stable localized secondary structures or decrease water solubility. In addition, AMOD includes an integrated multiple-species sequence comparison and host-speciﬁc genomic sequence val- idation and uniqueness assessment capabilities. The resulting output provides the user with a range of potential oligonuc- leotide designs suitable for use in a variety of biological applications, including the most common use as inhibitors of mRNA translation or for the alteration of pre-mRNA spli- cing. AMOD is a transparent, versatile and effective tool for short oligonucleotide and primer design. MATERIALS AND METHODS AMOD is written in PERL (http://www.perl.org/) and uses HTML and JavaScript for the user interface. BioPerl modules (20) are used for BLAST parsing and nucleotide-to-protein sequence translation. TIS predictions are made using the ATGpr web server (21). Sequence-to-sequence comparisons are performed using a local installation of NCBI BLAST ver- sion 2.1.2. Sequence comparisons may be made against ver- tebrate RefSeq proteins and the Ensembl zebraﬁsh genomic sequence set, housed in the Vertebrate Secretome and CTT- ome database (http://www.secretomes.umn.edu/). RESULTS AND DISCUSSION AMOD The AMOD design process consists of six steps separated into two phases, as shown in Figure 1. Phase one includes steps to characterize the nucleotide sequence and aid users in iden- tifying key sequence features, including the TIS and intron– exon splice sites. To facilitate the design of morpholinos for translational inhibition, TIS’s are predicted using ATGpr, a linear discriminate analysis program (21). Nucleotide sequences are automatically submitted to the ATGpr web ser- ver and predicted TISs ranked by prediction reliability scores, indices deﬁning the resulting open reading frame, and agree- ment with Kozak’s consensus sequence (22) is obtained. This program is used in AMOD because of its superior performance when analyzing expressed sequence tag (EST) sequences (23). To compliment this analysis, AMOD also performs an internal scan for ATG start codons. The scan identiﬁes all occurrences of the ‘ATG’ codon, the corresponding reading frame, assesses agreement to Kozak’s consensus sequence and identiﬁes the closest in-frame, upstream stop codon. Putative translational start sites identiﬁed by either method can be selected, and the TIS position highlighted in a display of the target gene sequence (Figure 2B). Homology modeling is used to further characterize the nucleotide sequences and conﬁrm the location of the TIS. Figure 1. Process flow chart for AMOD. The program consists of six steps, encompassing two phases: target sequence characterization and morpholino selection. Homology modeling is also useful for the identiﬁcation of splice junctions for splice site morpholinos. Provided that 25 bases on either side of the splice junction can be identiﬁed, other parameters for the design of splice site morpholinos are either similar to TIS-targeted morpholinos or yet to be determined. Users may execute sequence comparisons against RefSeq protein sequences or against a user supplied reference sequence set on the AMOD server. Users can also choose to upload an output ﬁle from a previously executed BLAST comparison, provided the output is in text format and the target nucleotide sequences were from the BLAST query sequences. The 10 highest scoring alignments for a gene sequence are presented in a BLAST report and alignment summary (Figure 2A). For blastx comparisons, N-terminal alignment extensions are dynamically constructed to aid users in assess- ing the target gene’s 50 completeness, using methods described previously (24). Phase two of the AMOD process entails the selection of a speciﬁc morpholino oligonucleotide, within the deﬁned gene subsequence possessing biochemical properties to improve morpholino water solubility and efﬁcacy as pioneered by Gene Tools. These considerations are the same for mor- pholinos targeting both TIS as well as splice junctions. AMOD creates an exhaustive list of putative morpholino oligonuc- leotides 25 bases long; the oligonucleotide length may be modiﬁed but is set to the size considered to result in optimal morpholino performance (25,26). For each morpholino, the G%, C%, A%, GC%, maximum number of consecutive adenines and maximum number of consecutive guanines in the oligonucleotide are calculated. Morpholino intra-strand