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Overview
What is Annotation?
Annotation is the process of determining the location and function of all identifiable genes in a genome.
Annotation is an important part of bioinformatics
• whole-genome shotgun sequencing provides the raw material
• annotation provides an interpretation of the sequencing results
Figure 1 from Stothard & Wishart (2006) Automated bacterial genome analysis and annotation. Current Opinion in Microbiology 9: 505-510.
1. Verify predicted function basedon amino acid sequence homology
2 Predict protein structure and localization
1. Find start and stop codons – separated by 800-900 bp?
2. Find Shine-Dalgarno sequence (RBS)– upstream of start codon?
3. Find core promoter – consensus sequences for -10 & -35?
4. Find rho-independent terminator
5. Predict whether the gene could beorganized into an operon– compare chromosomal neighborhood
What will we be doing?
Verifying ORF calls Verifying function based on sequence conservation
Verifying function based on structural conservation
Verifying function based on localization data
(insert image of E. coli lac permease)
Insert Figure 8-40 fromMicrobiology – An Evolving Science
© 2009 W.W. Norton & Company, Inc.
Why manually annotate?
• Automated annotations tend to over-predict….produce many false-positives
• Automated annotations also miss things….
• Accuracy of any annotation is only as good as the quality of annotated genes in reference databases
• High sequencing error rates. . .
A curated, finished genome has gene callsverified & proteins organized into pathways
Undergraduates provide “human expertise”
GOAL: Demonstrate that student annotationscan be accurate, up-to-date, reliable, and useful to scientific community!
Possible solutions?
Reference paper:Genome re-annotation: a wiki solution?by Steven SaltzbergGenome Biology (2007), 8:102
What is imgACT?
- Web portal to access genome database, img/edu
- Contains wiki-based Lab Notebook & Report Page for organizing annotation data
http://img-act.jgi-psf.org/user/login
http://imgweb.jgi-psf.org/cgi-bin/img_edu_v260/main.cgi
What is img/edu?
- Simplified database for undergraduate genome annotation
- Features and functions similar to that found in IMG
- Directly linked to imgACT
Click!
IMG companionsystem
http://img.jgi.doe.gov/cgi-bin/pub/main.cgi
What is IMG?
INTEGRATED MICROBIAL GENOMES (IMG)
- Database managed by the U.S. Department of Energy (DOE)Joint Genome Institute (JGI)
- JGI currently producing ~ 22% of the reported number of bacterial genome projects worldwide
- Key mission of IMG is to provide a data management platform that supports comprehensive analysis and annotation of all publicly available genomes in a comparative genomics context
What are we annotating?
(insert information about organism including location/map of collection site,image and description of organism, etc.)
Why annotate a GEBA organism?
Phylogenetic tree Phylogenetic tree of of BacteriaBacteria showing showing
established & established & candidate phylacandidate phyla
Insert Figure 1 from Handelsman (2004) Microbiol. Mol. Biol. Rev. 68: 669-685.
Note that genome sequences from members of those phyla in yellow and orange are under-represented relative to those in red
GEBA (Genomic Encyclopedia of Bacteria and Archaea) goal is to sequence genomes from under- represented phyla
What is our goal?
Insert Figure 2 from Scott KM et al. (2006) The Genome of Deep-Sea Vent Chemolithoautotroph Thiomicrospira crunogena XCL-2. PLoS Biology, 4: 2196
Annotate genes in pathways & complexes
Student Goals: Conceptual
• Apply basic concepts in biochemistry, microbial physiology & ecology, and evolutionary biology
• Question basic assumptions about biochemistry, physiology and evolution
• Understand the power and limitations of bioinformatics
• Proficiently use multiple database analysis software packages
• Strengthen web-based library search skills (Pubmed)
• Develop skills creating hypotheses and designing experiments to test them
• Sharpen skills in analysis, synthesis and presentation of results and data interpretation
• Experience the collaborative nature of science
Student Goals: Technical
• Each team will annotate genes encoding enzymes in a metabolic pathway or components of a cellular complex in [insert organism name]
• Your T.A. or instructor will tell you specific assignments
• Consult KEGG map and use orthologous gene in other related organisms to query the genome of [insert organism name] in IMG/EDU database
• For best “hit”, complete the corresponding modules of imgACT lab notebook and lab report for that gene
• Complete the module(s) presented each week. The imgACT online notebook & report for Modules #1 – 8 must be finished for all genes assigned (3 per student).
Annotation Project
Assignments
• Online notebook checks end of weeks:
• Final Report due dates:
Annotation Project
Click “Create an account”
http://img-act.jgi-psf.org/user/login
How do we get started?
Email address
First Name
Pick something youcan remember
Specific for our class
Click “Register” once information entered
Register for an img-act account
Last Name
Noabbreviationsor nicknames
xxxxxxxxxxxxxxxxxxxxxxxxxx
Once registration complete, log in to imgACT
What you should see. . .
If you can’t get this far, tell your instructor immediately!
Winter 2010
Next, take pre-annotation surveyCookies must be enabled for survey to work properly.
What next? Practice!
Explore the Explore the imgACTimgACT web portal web portal
• All students will be assigned at least one gene, which should be used to navigate through the imgACT online lab notebook (Modules #1 – 8) and the lab report
• Note that students are not responsible for annotating this gene. It may be used to help students get used to navigating the web portal.
“Practice gene”
click
click
imgACT Lab Notebook
The first time you log in to Lab Notebook, you will also need to log in to the wiki.Use the same username & password as created for imgACT account.
imgACT Lab Notebook
Only responsible for Modules #1 – 8 in this class
imgACT Lab Report
Correspond tomodules in
Lab Notebook
To be completedat end of the quarter