1. Welcome to DNA Subway Classroom-friendly Bioinformatics

2. DNA Subway Commonly used bioinformatics tools in streamlined workflows Teach important concepts in biology and bioinformatics Inquiry-based experiments for novel discovery and publication of data

3. DNA Subway Red Line: Genome annotation Red Line Analyze up to 150 KB of DNA sequence De novo gene prediction Construct evidence-based gene models Visualize genome sequence in browser

4. DNA Subway Yellow Line: Genome prospecting Yellow Line Analyze DNA or protein sequence Search plant genomes using TARGeT Explore gene duplications, transposons, and non-coding sequences not detectable in conventional BLAST searches

5. DNA Subway Blue Line: DNA barcoding, and phylogenetics Blue Line Analyze DNA or protein sequence Analyze DNA Barcoding sequence to identify plant, animal, and fungal species Generate phylogenetic trees and publish sequence to GenBank

6. DNA Subway Green Line: Transcriptome analysis Blue Line Examine RNA-Seq data for differential expression Use High-performance computing to analyze complete datasets Generate lists of genes and fold-changes; add results to Red Line projects

7. Annotate Genome Sequence Detect Genes and Build Gene Models DNA Subway: Red Line

8. DNA Subway Red Line: Genome annotation Requires JAVA 6 or above www.java.com Check your web browser has java enabled

9. Log in to DNA Subway

10. Create a project Detect all the genes present Import data from BLAST results and visualize in local browser Construct a gene model Verify gene model at Phytozome Task: Analyze a ~3KB sequence from Chromosome 1 of A.thaliana DNA Subway Red Line: Demo analysis – determine a structure for an Arabidopsis gene

11. Create a Red Line project DNA Subway Red Line: Demo analysis – determine a structure for an Arabidopsis gene

12. DNA Subway Red Line: Genome annotation Red Line 1.Click the Red Square to begin a project 2.Choose Plant and select Dicotyledon 3.Select sample sequence Arabidopsis thaliana (mouse-ear cress) Chr1, 3.40kb 4.Name the project and click Continue

13. Detect genes in the project sequence DNA Subway Red Line: Demo analysis – determine a structure for an Arabidopsis gene

14. DNA Subway Red Line 5.Click Sequence to view the input sequence Red Line: Demo analysis – determine a structure for an Arabidopsis gene

15. DNA Subway Red Line 6.Click Repeat Masker 7.When View icon ( ) appears; click Repeat Masker again to examine results Red Line: Demo analysis – determine a structure for an Arabidopsis gene

16. DNA Subway Tip: Before gene prediction, RepeatMasker attempts to identify repetitive sequences such as low-complexity, simple repeat, AT/GC-rich, or several types of transposons. Results are presented in a table. The Attributes column describes what type of repeat was detected in the ‘description=‘ field AT-rich sequence at 1667bps Red Line: Demo analysis – determine a structure for an Arabidopsis gene

17. DNA Subway Red Line 8.Click 1 or more gene predictors (Augustus, FGenesH, SNAP, tRNA Scan) 9.When View icon appears, click the gene predictor again to examine the results Red Line: Demo analysis – determine a structure for an Arabidopsis gene

18. DNA Subway Tip: de novo gene predictors predict genes within a given sequence. Each program is optimized differently; each program’s results vary. The Attributes column details features that make up a single predicted gene (e.g. the whole gene, mRNA, CDS, and exons). Sub-features are listed in the Type column. Red Line: Demo analysis – determine a structure for an Arabidopsis gene Augustus predicts a single gene (designated ‘g1’) with 4 exons

19. Import data from BLAST results and visualize in local browser DNA Subway Red Line: Demo analysis – determine a structure for an Arabidopsis gene

20. DNA Subway Red Line: Demo analysis – determine a structure for an Arabidopsis gene Red Line 10.Click BLASTN to search and import similar DNA sequences 11.Click BLASTX to search and import similar sequences based on protein evidence 12.When the searches complete; click again to examine results

21. DNA Subway Tip: BLAST results are derived from UNIGENE or UNIPROT databases, and contain experimentally derived evidence (e.g. cDNAs) that can be used to infer a probable gene structure. The Attributes column has details on the sequence matches that were found (e.g. gene name, GenBank IDs, etc.) Red Line: Demo analysis – determine a structure for an Arabidopsis gene

22. DNA Subway Red Line: Demo analysis – determine a structure for an Arabidopsis gene Red Line 13. Click Local Browser to visualize results

23. DNA Subway Tip: You can use the local browser (Gbrowse) at any time to visualize the results of any tool’s output. Red Line: Demo analysis – determine a structure for an Arabidopsis gene

24. Construct a gene model DNA Subway Red Line: Demo analysis – determine a structure for an Arabidopsis gene

25. DNA Subway Red Line: Demo analysis – determine a structure for an Arabidopsis gene Red Line 14. Click on Apollo to start the program

26. DNA Subway Red Line: Demo analysis – determine a structure for an Arabidopsis gene Red Line 15. Hide the reverse strand; click the View menu and select Hide Reverse Strand 16.Expand tiers; click the Tiers menu and select Expand all tiers 17.If there are too many tiers displayed, click the Tiers menu; select Show Types Panel and uncheck Show evidence you wish to hide

27. DNA Subway Red Line: Demo analysis – determine a structure for an Arabidopsis gene Red Line 18. Double-click the Augustus model and drag into workspace 19.Double-click the new temporary model; right-click to open the Annotation info editor 20. Name the model ‘Augustus1’ in both ‘Symbol’ fields.

28. DNA Subway Red Line: Demo analysis – determine a structure for an Arabidopsis gene Red Line 21. Double-click the BLASTN model and drag into workspace 22.Double-click the new temporary model; right-click to open the Annotation info editor 23. Name the model ‘BLASTN1’ in both ‘Symbol’ fields.

29. DNA Subway Red Line: Demo analysis – determine a structure for an Arabidopsis gene Red Line 23. Zoom in to examine the 5’ and 3’ ends of the gene models 24.Double-click the Augustus1 model and right-click to open the Exon detail editor 25. Adjust the 5’ and 3’ of the Augustus1 model to match the evidence provided by the BLASTN1 model

30. DNA Subway Tip: The BLASTN evidence is most useful for determining the transcript length (e.g. the 5’ and 3’ ends). Red Line: Demo analysis – determine a structure for an Arabidopsis gene

31. DNA Subway Red Line: Demo analysis – determine a structure for an Arabidopsis gene Red Line 26. Use any other available evidence* (e.g. BLASTN, User BLAST(N/X) ) to make alternative models if supported 27. Use the BLASTX evidence to determine start/stop codons. Drag any needed stop and start codon into your model. *If you have hidden evidence, show it again from the show types panel in the Tiers menu

32. DNA Subway Red Line: Demo analysis – determine a structure for an Arabidopsis gene Red Line 28.Delete the BLASTN1 model and any other extraneous models 29.Save your work back to DNA Subway; click the File menu and select Upload to DNA Subway; close Apollo

33. Verify gene model at Phytozome DNA Subway Red Line: Demo analysis – determine a structure for an Arabidopsis gene

34. DNA Subway Red Line: Demo analysis – determine a structure for an Arabidopsis gene Red Line 30.Click Phytozome Browser and compare the created model(s) to the accepted transcript(s)

35. DNA Subway Tip: Phytozome accepted transcripts are only available for DNA Subway sample sequences. Red Line: Demo analysis – determine a structure for an Arabidopsis gene