W510 Nucleic Acids Research, 2005, Vol. 33, Web Server issue guide the target sequence selection and morpholino design. In spite of this success, the analysis of relationships between phenotypic outcomes and sequence characteristics of mor- pholinos used in this screen have only identiﬁed a modest correlation between morpholino effectiveness and a minimum base composition threshold that was subsequently integrated into AMOD. No other improvement in parameters for mor- pholino design based on target gene sequence was strongly suggested by these outcomes. In addition, multiple factors arising from the use of a continually evolving zebraﬁsh genomic sequence database and incomplete EST sequence data limit the ability to discern speciﬁc guidelines for morpholino design or to evaluate the capacity of AMOD to improve morpholino design from this initial functional genom- ics screen. The exhaustive, robust evaluation of morpholino design parameters and programs for morpholino design await the identiﬁcation of a controlled reference set, including both efﬁcacious and non-efﬁcacious morpholinos. Until then, AMOD addresses the need to streamline target sequence selec- tion and morpholino design, e.g. in support of high-throughput approaches for morpholino screening. Comparison with other morpholino design services The sole commercial supplier of morpholinos, Gene Tools, offers a free, proprietary design service (http://www. gene-tools.com/). To our knowledge, this is presently the only other morpholino design service, requires prior user know- ledge of the translational start codon in the selected target mRNA, and provides very limited sequence design and analysis options. Based on the success of this proprietary method for morpholino design, AMOD was developed to implement sim- ilar morpholino design guidelines, such as avoidance of nuc- leotide motifs that form stable localized secondary structures or decrease water solubility. In an effort to improve on these initial design parameters and to incorporate the advancing genomic and transcriptome databases into morpholino design, AMOD includes an integrated multiple-species sequence comparison and host-speciﬁc genomic sequence validation and uniqueness assessment capabilities. The resulting output provides the user with a range of potential oligonucleotide designs suitable for use in a variety of biological applications, including their most common use as inhibitors of mRNA translation or for the altera- tion of pre-mRNA splicing. AMOD thus provides the user with a transparent design tool offering an increased number of oli- gonucleotide design options while implementing genomics- wide sequence criteria not found in the other available mor- pholino design option. AVAILABILITY AMOD web server is freely accessible to academic and com- mercial users at http://www.secretomes.umn.edu/AMOD/. A graphical step-by-step tutorial is available. For questions, comments and suggestions pertaining to AMOD, there is a link to an online ‘Contact Us’ form on every web page. FUTURE DEVELOPMENTS AND CONCLUSIONS The AMOD web server is a tool designed for selecting oligo- nucleotides for the construction of antisense morpholinos against target nucleotide sequences. Planned additions to the server include intron–exon and exon–intron splice site predictions, entropy calculations and a ranking system for putative morpholino designs. ACKNOWLEDGEMENTS The authors thank Michelle Knowlton, Aidas Nasevicius and Eric Mendenhall, for providing valuable feedback on AMOD’s function and user interface. Vanessa Louis, 2004 University of Minnesota undergraduate Bioinformatics Summer Intern (NSF/NIBIB EEC-0234112), assisted with analysis of relation- ships between phenotypic outcomes and sequence character- istics. This work was supported in part by NIH R01-GM63904, a predoctoral traineeship to E.K. (NLM TG-0704l), and a postdoctoral traineeship to M.A.P. (NIDCR: T32 DE07288-8). Funding to pay the Open Access publication charges for this article was provided by NIH R01-GM 63904. Conflict of interest statement. None declared. REFERENCES 1. Kos,R., Reedy,M.V., Johnson,R.L. and Erickson,C.A. (2001) The wingedhelix transcription factor FoxD3 is important for establishing the neural crest lineage and repressing melanogenesis in avian embryos. Development, 128, 1467–1479. 2. 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