BMC Genomics 2008, 9(Suppl 2):S20 http://www.biomedcentral.com/1471-2164/9/S2/S20 IFlliugsutrraeti2on of the steps in the BALBOA ORF classification algorithm Illustration of the steps in the BALBOA ORF classification algorithm. This figure shows the various step of the BAL- BOA ORF prediction algorithm. In step 1 the expression dataset is divided into its annotated genes and unannotated (unclassi- fied) ORFs. In step 2 biclusters are generated in the annotated gene set only. In step 3 selected biclusters (where E ≥ Emax) are used to classify similarly expressed ORFs in the unclassified set. In step 4 ORFs are combined into weighted frequency list for each functional category. Each ORF label weight is derived from the functional enrichments of the classifying biclusters. In step 5 the top ORFs (where F ≥ Fmax) are selected from this list. In step 6 ORFs consistently classified across independent datasets are returned. The full BALBOA strategy is illustrated in Figure 2. In step 1 the gene expression dataset is partitioned into annotated (genes with known functional labels) and unannotated (unclassified ORFs) subsets. In step 2 biclusters are then generated in the unannotated subset only. In step 3 biclus- ters that have relatively high functional enrichments, E, i.e. with E ≥ αEmax, where α is user defined and Emax is the maximum functional enrichment for a functional cate- gory, are used to assign labels to similarly expressed unclassified ORFs in the unannotated subset. In practice unclassified ORF profiles are first standardized (divided by their standard deviation). They are then sorted according to their mean squared residue score rela- tive to the bicluster classifier. This relative profile scoring was first used by Cheng and Church in their node deletion approach. In this case, the stopping point for the annota- tion process is determined retrospectively by the largest 'jump' in dissimilarity that occurs as the ordered ORFs are presented to the classifier. Page 6 of 15 (page number not for citation purposes)