1. Overview

2. 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

3. Figure 1 from Stothard & Wishart (2006) Automated bacterial genome analysis and annotation. Current Opinion in Microbiology 9: 505-510. 1.Verify predicted function based on 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 be organized into an operon – compare chromosomal neighborhood

4. 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 from Microbiology – An Evolving Science © 2009 W.W. Norton & Company, Inc.

5. 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 calls verified & proteins organized into pathways

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

7. 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

8. 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 companion system

9. 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

10. What are we annotating? (insert information about organism including location/map of collection site, image and description of organism, etc.)

11. Why annotate a GEBA organism? Phylogenetic tree of Bacteria showing established & candidate 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

12. 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

13. 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

14. 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

15. 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

16. Assignments Online notebook checks end of weeks: Final Report due dates: Annotation Project

17. Click “Create an account” http://img-act.jgi-psf.org/user/login How do we get started?

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

19. Once registration complete, log in to imgACT

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

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

22. What next? Practice! 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”

23. click

24. 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.

25. imgACT Lab Notebook Only responsible for Modules #1 – 8 in this class

26. imgACT Lab Report Correspond to modules in Lab Notebook To be completed at end of the quarter