Alternative transcription start site annotation for the example gene tramtrack. Flybase annotation of TSSs at the tramtrack locus of telease 4.3 . The gene span, Flybase mRNA, EST, and cDNA alignments were created using Gbrowse in Flybase . The locations of the EPD sites, hierarchically clustered TSSs, and start codon were added manually. There were three peaked TSSs listed in Flybase at locations 27539606 (TSS#1), 27550731 (TSS#2), and 27551187 (TSS#3). A fourth site at position 27552854 was listed, and is not shown, as it corresponded to the first nucleotide of the exon containing the start codon across all transcripts, and is likely to be an annotation artifact. The first TSS in EPD, EP77044, is 2 bp downstream of the Flybase TSS#2 at location 27550733. The second TSS, EP77045, occurred at location 27551504, and is 317 bp downstream of Flybase TSS#3. The distributions of ESTs at both locations were classified as single initiation sites by EPD on account of their high frequency and small dispersion. In the hierarchically clustered set, we observed TSSs at locations 27539771 (TSS#1), 27550733 (TSS#2), and 27551504 (TSS#3). The two most downstream TSSs correspond to the TSSs in EPD, and the most upstream TSS is close to the first TSS annotated in Flybase, but missing in EPD. This agreement with EPD resulted from our use of a similar dataset and identification strategy. All three Flybase TSSs for tramtrack are upstream of TSSs in the EPD and our sets, highlighting the bias in the usage of the most 5' evidence as TSSs, rather than the most highly utilized locations. Looking at the presence of sequence motifs within tramtrack peaked promoters, an INR was present at both TSS#1 and TSS#3 as defined in our set, strengthening our assignments for these TSSs, in spite of their considerably different locations in Flybase.