1. NGS Bioinformatics Workshop 1.5 Tutorial – Genome Annotation April 5th, 2012 IRMACS 10900 Facilitator: Richard Bruskiewich Adjunct Professor, MBB

2. Workflow for Today  Prepare to visualize annotation  Get a genomic sequence from Genbank  Repeat mask it.

3. Retrieve a genomic sequence…  Retrieve a (relatively small <100kb, eukaryote) genomic sequence clone from Genbank  Query Nucleotide divisione.g. Arabidopsis BAC clone (HE601748.1)  Select FASTA  Save.. To File.. As “Fasta” (rename?)

4. Blast is a low hanging fruit…  Use BLAST to quickly survey for similar sequences  Megablast against nucleotide e.g. HE601748 is closest to A. thaliana chr. 5?  Megablast against reference RNA sequence db

5. Repeat Masking  Upload the clone file to RepeatMasker on the web and run with appropriate parameters: http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker  Save the results (including the masked sequence) to your computer

6. ab initio Gene Predictions  Genscan: http://genes.mit.edu/GENSCAN.html  Cut and paste results as text to a file  Fgenesh: www.softberry.com

7. Blast2GO http://www.blast2go.com  Annotation workbench, via Gene Ontology (GO) terms.  First, save the predicted peptides (e.g. from fgenesh)  need to fix the FASTA headers to assign proper identifiers (could write a script?)  (Java web) start blast2go workbench  Load in peptides  Do the analysis… e.g. run blastp, GO, annotation, Interpro, etc.  See www.geneontology.org for details on GOwww.geneontology.org  http://www.ebi.ac.uk/interpro/ for interpro info http://www.ebi.ac.uk/interpro/

8. EMBOSS  European Molecular Biology Open Software Suite (EMBOSS): http://emboss.sourceforge.net  Download and install version of interest (e.g. Linux, Mac OSX, Windows…)  Decide what do to: http://emboss.sourceforge.net/apps/groups.html  Let’s try a CpG island plot (cpgplot)

9. Study Genes by Comparative Genomics  JGI Vista toolkit:  http://genome.lbl.gov/vista http://genome.lbl.gov/vista  GenomeVista  rVista