Subsystem Approach to Genome Annotation

1. Subsystem Approach to Genome Annotation National Microbial Pathogen Data Resource www.nmpdr.org Claudia Reich NCSA, University of Illinois, Urbana

2. Complete Microbial Genomes • 464 complete microbial genomes in NCBI as of 3-1-07 • 691 microbial genomes in progress as of 3-1-07 www.nmpdr.org

3. Making Sense of Genome Data • Locate Genes: identify ORFs automatically • GeneMark • NCBI’s ORF Finder • Glimmer • Critica • Assign Function: by sequence similarity to experimentally characterized proteins • BLAST family of sequence comparison tools www.nmpdr.org

4. Problems with Assignments by Similarity • When ORF is a member of a protein family • Paralogous genes • ORFs encoding similar proteins acting on different substrates • Assignments can be transitive, and many times removed from experimental data www.nmpdr.org

5. Other Factors Can Aid in Function Assignments • Molecular phylogeny • Paralogous and orthologous families • Conserved gene neighborhood • Metabolic context • Bidirectional best hit matches across multiple genomes www.nmpdr.org

6. Incorporating Information Other Than Similarity • KEGG: manually curated pathway and metabolic maps • GO: vocabularies that describe ORFs as associated with • biological processes • cellular components • molecular function • MetaCyc: experimentally elucidated metabolic pathways www.nmpdr.org

7. What is Needed: • A system that: • integrates all the above concepts • organizes genomic data in structured idioms • allows high-throughput annotation of newly sequenced genomes • resolves discrepancies in different annotation tools • informs experimental research www.nmpdr.org

8. Enter the SEED* • Database and annotation environment • Underlies, and accessible through, NMPDR (www.nmpdr.org) • Expert annotation via subsystems building • Provides the most accurate genome annotations available *Argonne National Lab, University of Chicago, UIUC, FIG www.nmpdr.org

9. What is a Subsystem? • Any organizing biological principle: • metabolic pathway • amino acid biosynthesis, nitrogen fixation, glycolysis • complex structure • ribosome, flagellum • set of defining features • virulome, pathogenicity islands • functional concept • bacterial sigma factors, DNA binding proteins www.nmpdr.org

10. Subsystems are: • Sets of functional roles, which are functions, or abstractions of functions (such as an EC number), that together implement a specific biological process or concept • Created manually by expert curators • Experts annotate single subsystems over the complete collection of genomes, thus contributing and sharing their expertise with the scientific community www.nmpdr.org

11. How Subsystems are Built • Create a subsystem for the biological concept, and define the functional roles • In one (or a few) key organisms that include the subsystem, find the genes and assign meaningful functional names • Project the annotations to orthologous genes • Expand to more genomes, creating a Populated Subsystem www.nmpdr.org

12. Populated Subsystems • Are Spreadsheets where: • Columns: functional roles • Rows: specific genomes • Cells: genes in the organism that implement the functional role www.nmpdr.org

13. How to Access Subsystems • From Home page (left navigation bar): Subsystem Summaries: select organism • From Organism pages • From Subsystem Search • From protein pages: to specific subsystems www.nmpdr.org

14. Subsystem Pages in NMPDR • Table of Functional Roles • Subsystem diagram (if appropriate) • Populated subsystem spreadsheet • Customizable spreadsheet viewing options • Functional variants and subsets of roles • Curator’s notes www.nmpdr.org

15. Benefits of Subsystems • More accurate annotations • Annotation of protein families • Analysis of sets of functionally related proteins • Less error-prone to automatic projections to novel genomes www.nmpdr.org

16. Subsystems Reveal Interesting • Pathway variants: • Are they clustered by phylogeny? • Delta subunit of RNA polymerase only Bacillales • Are they clustered by functional niche? • Horizontal gene transfer? • Fused genes: •  and ’ subunit of RNA polymerase fused in Helicobacter • Fissioned genes: • ’ subunit of RNA polymerase is fissioned in Cyanobacteria www.nmpdr.org

17. Subsystems Reveal Interesting • Duplicate assignments • More than one gene for one functional role? • Alpha subunit of RNA polymerase in Magnetococcus and Francisella • Same sequenced region in more than one contig in partially assembled genomes? • Frameshifts or other sequencing errors? • Annotation errors? www.nmpdr.org

18. Subsystems Reveal Interesting • Missing genes: • Is the function essential? • Is the function conserved? • Does the missing gene cluster with homologs in other organisms? • Is the function performed by a newly recruited gene? • Has a gene been acquired by horizontal gene transfer and now performs that function? www.nmpdr.org

19. Synthesis of Selenocysteinyl-tRNA • Two known pathway variants • One step in Bacteria • SelA is annotated • Two steps in Archaea and Eucarya • PSTK was missing until very recently www.nmpdr.org

20. Explore Selenocysteine Usage • Start by searching for gene name, selA, in an organism known to use Sec, E. coli K12 • Start from subsystem tree; expand category of "Protein metabolism," expand subcategory of "Selenoproteins" • Open "Selenocysteine metabolism" subsystem from protein page or SS tree • Genomes arranged phylogenetically • Roles defined on mouse-over • What genes are missing in which organisms? • Are there Sec metabolism genes present in any organisms that do not have proteins that need Sec? • Are there organisms known to need Sec for certain proteins, but that do not have a complete Sec biosynthesis pathway? • Why is there a hypothetical protein included in this subsystem? www.nmpdr.org