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NGS Bioinformatics Workshop 1.5 Tutorial – Genome Annotation. April 5th, 2012 IRMACS 10900 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….

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ngs bioinformatics workshop 1 5 tutorial genome annotation

NGS Bioinformatics Workshop1.5 Tutorial – Genome Annotation

April 5th, 2012

IRMACS 10900

Facilitator: Richard Bruskiewich

Adjunct Professor, MBB

workflow for today
Workflow for Today
  • Prepare to visualize annotation
  • Get a genomic sequence from Genbank
  • Repeat mask it.
retrieve a genomic sequence
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?)
blast is a low hanging fruit
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
repeat masking
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
ab initio gene predictions
ab initio Gene Predictions
  • Genscan:

http://genes.mit.edu/GENSCAN.html

    • Cut and paste results as text to a file
  • Fgenesh:

www.softberry.com

blast2go
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 GO
  • http://www.ebi.ac.uk/interpro/ for interpro info
emboss
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)
study genes by comparative genomics
Study Genes by Comparative Genomics
  • JGI Vista toolkit:
  • http://genome.lbl.gov/vista
  • GenomeVista
  • rVista