1. 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:

2. 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”