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

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

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

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

Repeat Masking  Upload the clone file to RepeatMasker on the web and run with appropriate parameters:  Save the results (including the masked sequence) to your computer

ab initio Gene Predictions  Genscan:  Cut and paste results as text to a file  Fgenesh:

Blast2GO  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 for details on GOwww.geneontology.org  for interpro info

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

Study Genes by Comparative Genomics  JGI Vista toolkit:   GenomeVista  rVista