1. Web Apollo Resources at the National Agricultural Library Christopher Childers NAL ARS USDA i5k.nal.usda.gov

2. Agenda Web Apollo – Web based tool for manual gene curation – Registration required for access Introduction to the Web Apollo interface – Viewing evidence tracks – Logging in/out BLAST Example annotation

3. Annotation Overview 1.Find sequence 2.Align to genome 3.Examine available evidence 4.Annotate based on evidence 5.Compare results to related sequences – BLAST/BLAT or multiple alignment 6.Repeat steps 1 through 5 as needed to refine model 7.Add annotation details in the “Information Editor”

4. Annotator Registration

5. Some conventions HSP – High scoring pair in BLAST/BLAT alignments – The ‘Hits’ in an alignment result set – A subsection of a pair of sequences with sufficient score – Change based on the alignment parameters Five prime end and three prime end – Based on direction of transcription – Initiation site is at the five prime end – Stop codon is at the three prime end In the genome browser, arrowheads indicate direction 3’ 5’ 5’ 3’

6. Web Apollo/JBrowse JBrowse is a web based genome browser – Visualize features that are mapped to a genome – These features are displayed as tracks – Many different types of data may be Web Apollo adds editing functions to JBrowse – Manual gene curation – Changes automatically saved back to server – Edits are visible to other annotators in real-time

7. Web Apollo 1 3 2

8. Viewing evidence 1.The “Available Tracks” panel lists all tracks – Tracks are grouped hierarchically Study or analysis Categories based on evidence type 2.The Navigation panel describes the viewed region – Coordinate ranges for the entire sequence on top – Navigation controls including pan, zoom and search in the middle – Coordinate ranges for the current view along the bottom 3.The Evidence panel shows the features in genomic context – Each type of data is a separate track – Tracks may be displayed in different ways

9. Viewing evidence Turning tracks on or off – Click a tracks checkbox to turn it on or off – Click ‘X’ by the track label also turns the track off

10. Login For scaffold selection view:

11. Login Directly from JBrowse genome browser

12. Login Directly from JBrowse genome browser

13. Login Directly from JBrowse genome browser

14. BLAST at the i5k Workspace Redesigned: uses the latest version of BLAST+ Interface updates options automatically – Based on the combination of query and database – Chooses correct options (blastn, blastx, tblastn, etc) Results persist for one week – The results URL can be saved or shared HSPs are viewable in the genome browser – HSPs are conservatively chained together – Visible as connected blocks

15. BLAST

16. BLAST Results

17. BLAST results in Web Apollo

18. Live Annotation Example Alpha-catenin in the Colorado Potato Beetle We created special training versions of our tools BLAST: https://i5k.nal.usda.gov/training/blast Web Apollo: https://apollo.nal.usda.gov/lepdec_training/jbrowse

22. Annotation using real data Logging in to the demo instance Possible gene families to look at – Cytochrome P450 – myotubularin-related proteins – COP9

23. Sequence Search Map a gene of interest to a genomic region – BLAT: Web Apollo plugin – BLAST@NAL: separate tool Each has unique features – BLAT shows the hit as a highlighted region – BLAST shows hits as segments, similar to other evidence tracks