Experimental evidence for S. cerevisiae orphan ORFs. (A) Percentages indicate proportions of orphan ORFs detected at least in one of 13 functional genomics and proteomics data sets (Table 1). Note that ORFs rejected by all three comparative genomic studies analyzed here (Brachat et al. 2003; Cliften et al. 2003; Kellis et al. 2003) show similar percentages. (B) Supporting experimental evidence for each of 648 ORFs observed as orphan by three comparative genomic studies (Brachat et al. 2003; Cliften et al. 2003; Kellis et al. 2003). Complete lists of ORFs and supporting experimental evidence are in Supplemental Table 2. Columns are ordered from the ORF with most evidence (ORF X₁; left) to the one with the least evidence (ORF X₆₄₈; right). Data sets were grouped together by type of experimental approach, transcriptional on top and translational at the bottom. In total, there are 477 orphan ORFs with transcriptional evidence, 180 with translational evidence, and 145 with both transcriptional and translational evidence.

Two-step strand-specific RT-PCR. (A) Schematic diagram of the strand-specific RT-PCR procedure. (B) Electrophoretic analysis of strand-specific RT-PCR products. Reverse ORF-specific primers (OSP^R), with sequences complementary to the ORF-coding strand, were used for first-strand cDNA synthesis. Second-step PCR amplifications used a pair of forward (OSP^F) and reverse ORF-specific primers (OSP^R). As controls, the first step of RT-PCR was performed without reverse transcriptase for detecting contamination by genomic DNA, or without the OSP^R primer for detecting residual reverse transcriptase activity in second-step PCR reactions. Two intron-containing verified ORFs, YER133W (genomic DNA length: 1464 bp; coding sequence length: 939 bp) and YBR078W (genomic DNA length: 1737 bp; coding sequence length: 1407 bp), were used to test the strand specificity. An extra control for these two verified ORFs was a standard PCR action using yeast genomic DNA as template and the same pair of ORF-specific primers. The observed difference in the length of PCR products amplified from genomic DNA versus poly(A) mRNA manifested the strand specificity. Strand-specific RT-PCR results of 201 nonoverlapping orphan ORFs were analyzed on 1% agarose E-gel (Invitrogen). Of the reactions 53% (105 ORFs) gave rise to visible RT-PCR products of the expected sizes. Three orphan ORFs, YJL199C (327 bp), YJR108W (372 bp), and YDR344C (444 bp), are shown as examples of successful RT-PCR reactions. (C) Comparison of the average LLR between nonoverlapping ORFs detected and undetected by strand-specific RT-PCR. Error bars, SEM.

Evaluating functionality of S. cerevisiae ORFs. (A) ROC curve (blue) for naïve Bayes predictions based on 13 functional genomics and proteomics data sets. The diagonal (black dotted line) is the expected ROC curve for random, where the TP rate equals the FP rate. The two LLR cutoffs highlighted on the curve were used later as thresholds for categorizing orphan ORFs. (B) All 6718 S. cerevisiae ORFs were divided into 20 bins by decreasing LLR. Each bin has similar numbers of ORFs. The false-positive rates associated with the minimum LLR in each bin are listed. Distributions of verified ORFs, orphan dubious ORFs, “other” dubious ORFs, and all other ORFs in each bin are shown. Orphan dubious ORFs tend to have a higher LLR than ORFs classified as dubious for other reasons.

Interstrain intraspecies sequence conservation for orphan ORFs. (A) Distribution of nucleotide divergence in different genomic features. We binned three types of genomic features, (1) non-orphan ORFs (red curve), (2) orphan ORFs predicted by three comparative genomic analyses (blue curve) (Brachat et al. 2003; Cliften et al. 2003; Kellis et al. 2003), and (3) intergenic regions (green curve), using a window of an average three SNPs per kb across four S. cerevisiae strains. Each dot represents the fraction of genomic features in each bin. Numbers on the X-axis represent the maximum number of SNPs per kb in each bin. For instance the first bin collects the genomic regions that have between zero and three SNPs per kb in four strains. The inset zooms in on the 0–21 SNPs per kb range with SEM displayed. (B) Comparison of nucleotide divergence among three predicted categories of orphan ORFs based on their LLRs. Error bars, SEM in each category. (C) Comparison of the percentage of ORFs among the three predicted categories of orphan ORFs that have reading frames preserved across four S. cerevisiae strains.