D188 Nucleic Acids Research, 2008, Vol. 36, Database issue However, the pre-miRNA ebv-mir-BHRF1-2 has two mature miRNAs in its 50 arm and 30 arm. In addition, users may download the entire sequences of predicted candidate hairpins via the link at the bottom of each page. Besides, we also extracted all known viral miRNAs from miRBase into an independent table that also can be retrieved at http://alk.ibms.sinica.edu.tw/cgi-bin/miRNA/ known_viral_miRNA.cgi. Predicting potential viral miRNA target genes using the RNAhybrid program It is of researchers’ interest to learn about viral miRNAs and their host target genes. To assist biologists, we provide an integrated interface to identify possible host target genes of the viral miRNA in selected host genomes using the RNAhybrid program (19,20). Presently, as shown in Figure 4, we provided the 30-UTR region sequences of reference genes from human, mouse, rat, zebraﬁsh, rice and Arabidopsis as the search target dataset. We retrieved 30 UTRs based on the coding sequence positions acquired individually from the human.rna.gbﬀ ﬁle (from NCBI on 19 December 2005), mouse.rna.gbﬀ ﬁle (from NCBI on 19 December 2005), rat.rna.gbﬀ ﬁle (from NCBI on 19 December 2005), zebraﬁsh.rna.gbﬀ ﬁle (from NCBI on 19 June 2006) and plant1 5.rna.gbﬀ ﬁle (from NCBI on 5 August 2006). Users may scan the 30-UTR region sequences of reference genes for possible viral miRNA targets. When operating RNAhybrid, the pipeline ﬁrst calculates the optimal free energy of a putative miRNA when the entire putative miRNA binds to a perfectly complementary target site, then it calculates the minimum free energy of RNA duplex (mfe of the miRNA/mRNA duplex). An alignment for which the RNA duplex mfe is more than 66% of its correspondent optimal free energy is regarded as a positive alignment as described by Krek et al. (21). A higher stringent 85% parameter is recommend. A critical issue on the target prediction is that it dramatically depends on the seed region located in the mature miRNA. However, it is diﬃcult to correctly predict the mature miRNA following the hairpin predic- tion pipeline. Therefore, we have a helix constraint option to let users select diﬀerent seed match region to be used in the target prediction pipeline. It is suggested this information should be used for preliminary exploration purpose without the mature miRNA information. DISCUSSION The available predicted viral miRNA candidate hairpins will certainly be beneﬁcial to biologists who study interactions between viral miRNAs and host genes. There are many completed viral genome sequences in the database, but it is not a simple task for biologists to execute the entire bioinformatic pipeline to scan for putative miRNA hairpins. We have identiﬁed 33691 candidate hairpins from more than 2000 virus genome sequences. Here, we provide an easy-to-use web interface Figure 4. Target gene search web interface. Presently, we provide the 30-UTR region sequences of human, mouse, rat, zebraﬁsh, rice and Arabidopsis for target gene prediction. The RNAhybrid program is applied for the target search (19,20). Users can directly submit the predicted viral miRNA hairpin sequences from the viral miRNA web interface or submit their own miRNA sequences for target gene prediction. We have a helix constraint option to let users select diﬀerent seed match region to be used in the target prediction pipeline. Since it is diﬃcult to correctly predict the mature miRNA from the hairpin structure. It is suggested this target prediction information should be used for preliminary exploration purpose.