TSS Annotation Workflow Identify ortholog D. melanogaster Target species Characterize TSS in the D. melanogaster ortholog Identify isoforms with unique TSS FlyBase GBrowse 2 Gene Record Finder For each unique TSS GEP UCSC Genome Browser (D. melanogaster) Classify core promoter (peaked / intermediate / broad / insufficient evidence) 1. Transcription start sites (Celniker > Embryonic) 2. DNase I hypersensitive sites 3. 9-state model Annotate TSS in the target species (e.g., D. biarmipes) 1. blastn (sensitive parameters) 2. RNA-Seq coverage 3. TopHat junctions 4. Conservation track 5. Placements of all 5’ UTR exons 6. Core promoter motifs Annotate the initial transcribed exon Identify core promoter motifs* in the target species and in D. melanogaster (±300 bps from TSS and/or in narrow search region) GEP UCSC Genome Browser (target species) Short Match track (e.g., Inr) Define TSS positions or TSS narrow and wide search regions Synthesize the evidence: blastn > RNA-Seq > motifs * Drosophila core promoter motifs: http://gander.wustl.edu/~wilson/core_promoter_motifs.html

TSS Annotation Workflow (Recommended Parameters) Characterize TSS in the D. melanogaster ortholog GEP UCSC Genome Browser (D. melanogaster) Transcription start sites TSS (Celniker) (R5): pack DNase I hypersensitive sites Detected DHS Positions (Cell Lines) (R5): pack DHS Read Density (Cell Lines) (R5): full 9-state model BG3 9-state (R5): dense S2 9-state (R5): dense Types of core promoters Classification # TSS #DHS Peaked 1 Intermediate ≤ 1 > 1 Broad Annotate TSS in the target species Should we add more TSS search regions Annotate the initial transcribed exon Define TSS position(s) blastn search parameters Similarity to the initial transcribed exon in D. melanogaster (blastn) RNA-Seq and Conservation tracks Position(s) of core promoter motifs Word size: 7 Match / Mismatch scores: 1 / -1 Gap costs: Existence: 2, Extension: 1 Low complexity filter: off RNA-Seq tracks RNA-Seq Alignment Summary: show RNA-Seq TopHat: squish Comparative genomics tracks Additional resources Conservation: full Genome alignments of 26 Drosophila species RNA PolII ChIP-Seq tracks (D. biarmipes) N-SCAN and Augustus TSS predictions Core promoter motifs search 1. Click on the “Short Match” link 2. Enter the motif consensus sequence 3. Change the display mode to “pack” 4. Click “Submit”