Overview

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.
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 be organized into an operon
– compare chromosomal neighborhood
1.
Verify predicted function based on amino acid sequence homology
2 Predict protein structure and localization

Verifying ORF calls
Verifying function based on sequence conservation
Insert Figure 8-40 from
Microbiology – An Evolving Science
© 2009 W.W. Norton & Company, Inc.
Verifying function based on localization data
Verifying function based on structural conservation
(insert image of E. coli lac permease)

• 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 calls verified & proteins organized into pathways

Reference paper:
Genome re-annotation: a wiki solution?
by Steven Saltzberg
Genome Biology (2007), 8 :102
Undergraduates provide “human expertise”
GOAL: Demonstrate that student annotations can be accurate, up-to-date, reliable, and useful to scientific community!

http://img-act.jgi-psf.org/user/login
- Web portal to access genome database, img/edu
- Contains wiki-based Lab Notebook & Report Page for organizing annotation data

http://imgweb.jgi-psf.org/cgi-bin/img_edu_v260/main.cgi
- Simplified database for undergraduate genome annotation
- Features and functions similar to that found in IMG
- Directly linked to imgACT
Click!
IMG companion system

http://img.jgi.doe.gov/cgi-bin/pub/main.cgi
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

(insert information about organism including location/map of collection site, image and description of organism, etc.)

Phylogenetic tree of Bacteria showing established & candidate phyla
 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 underrepresented phyla
Insert Figure 1 from Handelsman (2004)
Microbiol. Mol. Biol. Rev . 68 : 669-685.

Annotate genes in pathways & complexes
Insert Figure 2 from Scott KM et al . (2006) The Genome of Deep-Sea Vent Chemolithoautotroph Thiomicrospira crunogena XCL-2. PLoS Biology , 4 : 2196

• 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

• 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).

Assignments
• Online notebook checks end of weeks:
• Final Report due dates:

http://img-act.jgi-psf.org/user/login
Click “Create an account”

Register for an img-act account
Email address
First Name
Last Name
No abbreviations or nicknames xxxxxxxxxxxxxxxxxxxxxxxxxx
Pick something you can remember
Specific for our class
Click “Register” once information entered

Once registration complete, log in to imgACT

What you should see. . .
Winter 2010
If you can’t get this far, tell your instructor immediately!

Next, take pre-annotation survey
Cookies must be enabled for survey to work properly.

Explore the imgACT 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
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.

Only responsible for Modules #1 – 8 in this class

To be completed at end of the quarter
Correspond to modules in
Lab Notebook