Pathway Bioinformatics: Inference, Visualization, and Analysis. Peter D. Karp, Ph.D.

Transcription

1 Pathway Bioinformatics: Inference, Visualization, and Analysis Peter D. Karp, Ph.D. Bioinformatics Research Group SRI International BioCyc.org EcoCyc.org, MetaCyc.org, HumanCyc.org 1 SRI International Bioinformatics

2 Overview Motivations Pathway/genome databases BioCyc collection EcoCyc, MetaCyc Pathway Tools software Visualization, Editing, Analysis Inference tools: Pathway hole filler, Transport inference parser Reachability analysis of metabolic network Applications New levels of genome annotation Omics data analysis Publish curated pathway/genome database on the web 2 SRI International Bioinformatics

3 Model Organism Databases / Organism Specific Databases DBs that describe the genome and other information about an organism Every sequenced organism with an active experimental community requires a MOD Integrate genome data with information about the biochemical and genetic network of the organism Integrate literature-based information with computational predictions Curated by experts for that organism No one group can curate all the world s genomes Distribute workload across a community of experts to create a community resource 3 SRI International Bioinformatics

4 Rationale for MODs Each complete genome is incomplete in several respects: 40%-60% of genes have no assigned function Roughly 7% of those assigned functions are incorrect Many assigned functions are non-specific Need continuous updating of annotations with respect to new experimental data and computational predictions Gene positions, sequence, gene functions, regulatory sites, pathways MODs are platforms for global analyses of an organism Interpret omics data in a pathway context In silico prediction of essential genes Characterize systems properties of metabolic and genetic networks 4 SRI International Bioinformatics

5 BioCyc Collection of Pathway/Genome Databases Pathway/Genome Database (PGDB) combines information about Pathways, reactions, substrates Enzymes, transporters Genes, replicons Transcription factors/sites, promoters, operons Tier 1: Literature-Derived PGDBs MetaCyc EcoCyc -- Escherichia coli K-12 Tier 2: Computationally-derived DBs, Some Curation PGDBs HumanCyc Mycobacterium tuberculosis Tier 3: Computationally-derived DBs, No Curation DBs 5 SRI International Bioinformatics

6 Pathway Tools Overview Annotated Genome PathoLogic MetaCyc Reference Pathway DB Pathway/Genome Database Pathway/Genome Editors Pathway/Genome Navigator 6 SRI International Bioinformatics

7 Pathway Tools Software: PathoLogic Computational creation of new Pathway/Genome Databases Transforms genome into Pathway Tools schema and layers inferred information above the genome Predicts operons Predicts metabolic network Predicts which genes code for missing enzymes in metabolic pathways Infers transport reactions from transporter names Bioinformatics 18:S SRI International Bioinformatics

8 Pathway Tools Software: Pathway/Genome Editors Interactively update PGDBs with graphical editors Support geographically distributed teams of curators with object database system Gene editor Protein editor Reaction editor Compound editor Pathway editor Operon editor Publication editor 8 SRI International Bioinformatics

9 Pathway Tools Software: Pathway/Genome Navigator Querying and visualization of: Pathways Reactions Metabolites Proteins Genes Chromosomes Two modes of operation: Web mode Desktop mode Most functionality shared, but each has unique functionality 9 SRI International Bioinformatics

10 Pathway Tools Ontology / Schema Ontology classes: 1621 Many datatypes from genomes to pathways Classification schemes for pathways, chemical compounds, enzymatic reactions (EC system) Cell Component Ontology Protein Feature ontology Comprehensive set of 221 attributes and relationships Evidence codes, supporting citations 10 SRI International Bioinformatics

11 BioCyc Tier 3 Tier 3 DBs created by subjecting annotated genomes to PathoLogic computational processing pipeline: Pathway prediction Pathway Hole Filling Operon prediction (bacteria) Transport Inference Parser 349 PGDBs Source: CMR database 11 SRI International Bioinformatics

12 Pathway Tools Software: PGDBs Created Outside SRI 1,300+ licensees: 75+ groups applying software to 200+ organisms Saccharomyces cerevisiae, SGD project, Stanford University Mouse, MGD, Jackson Laboratory dictybase, Northwestern University Under development: CGD (Candida albicans), Stanford University Drosophila, P. Ebert in collaboration with FlyBase C. elegans, P. Ebert in collaboration with WormBase Planned: RGD (Rat), Medical College of Wisconsin Arabidopsis thaliana, TAIR, Carnegie Institution of Washington PlantCyc, ~20 plant PGDBs, Carnegie Institution of Washington Six Solanaceae species, Cornell University GrameneDB, Cold Spring Harbor Laboratory Medicago truncatula, Samuel Roberts Noble Foundation 12 SRI International Bioinformatics

13 EcoCyc Project EcoCyc.org E. coli Encyclopedia Review-level Model-Organism Database for E. coli Tracks evolving annotation of the E. coli genome and cellular networks The two paradigms of EcoCyc Multi-dimensional annotation of the E. coli K-12 genome Positions of genes; functions of gene products 76% / 66% exp Gene Ontology terms; MultiFun terms Gene product summaries and literature citations Evidence codes Multimeric complexes Metabolic pathways Regulation of transcription initiation Karp, Gunsalus, Collado-Vides, Paulsen Nuc. Acids Res. 35: ASM News 70: Science 293: SRI International Bioinformatics

14 EcoCyc = E.coli Dataset + Pathway/Genome Navigator URL: EcoCyc.org Pathways: 224 Reactions: Metabolic: 923 Transport: 259 Compounds: 1,227 Citations: 16,153 Proteins: 4,464 Complexes: 808 RNAs: 285 Genes: 4,471 Gene Regulation: Operons: 3,187 Trans Factors: 179 Promoters: 1,343 TF Binding Sites: 1, SRI International Bioinformatics

15 Paradigm 1: EcoCyc as Textual Review Article All gene products for which experimental literature exists are curated with a minireview summary Found on protein and RNA pages, not gene pages! 3257 gene products contain summaries Summaries cover function, interactions, mutant phenotypes, crystal structures, regulation, and more Additional summaries found in pages for operons, pathways EcoCyc cites 15,880 publications 15 SRI International Bioinformatics

16 Paradigm 2: EcoCyc as Computational Symbolic Theory Highly structured, high-fidelity knowledge representation provides computable information Each molecular species defined as a DB object Genes, proteins, small molecules Each molecular interaction defined as a DB object Metabolic reactions Transport reactions Transcriptional regulation of gene expression 220 database fields capture extensive properties and relationships 16 SRI International Bioinformatics

17 EcoCyc Procedures DB updates performed by 5 staff curators Information gathered from biomedical literature Enter data into structured database fields Author extensive summaries Update evidence codes Corrections submitted by E. coli researchers Four releases per year Quality assurance of data and software Evaluate database consistency constraints Perform element balancing of reactions Run other checking programs 17 SRI International Bioinformatics

18 EcoCyc Accelerates Science Experimentalists E. coli experimentalists Experimentalists working with other microbes Analysis of expression data Computational biologists Biological research using computational methods Genome annotation Study connectivity of E. coli metabolic network Study phylogentic extent of metabolic pathways and enzymes in all domains of life Bioinformaticists Training and validation of new bioinformatics algorithms predict operons, promoters, protein functional linkages, protein-protein interactions, Metabolic engineers Design of organisms for the production of organic acids, amino acids, ethanol, hydrogen, and solvents Educators 18 SRI International Bioinformatics

19 MetaCyc: Metabolic Encyclopedia Describe a representative sample of every experimentally determined metabolic pathway Describe properties of metabolic enzymes Literature-based DB with extensive references and commentary Pathways, reactions, enzymes, substrates Jointly developed by P. Karp, R. Caspi, C. Fulcher, SRI International L. Mueller, A. Pujar, Cornell Univ S. Rhee, P. Zhang, Carnegie Institution Nucleic Acids Research SRI International Bioinformatics

20 Applications of MetaCyc Reference source on metabolic pathways Metabolic engineering Find enzymes with desired activities, regulatory properties Determine cofactor requirements Predict pathways from genomes Systematic studies of metabolism Computer-aided education 20 SRI International Bioinformatics

21 MetaCyc Data -- Version 12.0 Pathways Reactions Enzymes Small Molecules Organisms Citations ,739 4,731 6,719 1,108 16, SRI International Bioinformatics

22 Taxonomic Distribution of MetaCyc Pathways version 12.0 Bacteria Green Plants Mammals Fungi Archaea SRI International Bioinformatics

23 Family of Pathway/Genome Databases MetaCyc EcoCyc CauloCyc AraCyc MtbRvCyc HumanCyc 23 SRI International Bioinformatics

24 PathoLogic Step 1: Translate Genome to PGDB Annotated Genomic Sequence Gene Products Pathway/Genome Database Pathways Genes/ORFs DNA Sequences Multi-organism Pathway Database (MetaCyc) Pathways Reactions Compounds PathoLogic Software Integrates genome and pathway data to identify putative metabolic networks Reactions Compounds Gene Products Genes Genomic Map 24 SRI International Bioinformatics

25 PathoLogic Step 4: Pathway Hole Filler Definition: Pathway Holes are reactions in metabolic pathways for which no enzyme is identified L-aspartate iminoaspartate quinolinate synthetase nada quinolinate NAD+ synthetase, NH3 - dependent CC3619 holes deamido-nad n.n. pyrophosphorylase nadc nicotinate nucleotide NAD SRI International Bioinformatics

26 Step 1: Query UniProt for all sequences having EC# of pathway hole organism 1 enzyme A organism 2 enzyme A organism 3 enzyme A organism 4 enzyme A organism 5 enzyme A organism 6 enzyme A organism 7 enzyme A organism 8 enzyme A Step 2: BLAST against target genome gene X gene Y gene Z Step 3 & 4: Consolidate hits and evaluate evidence 7 queries have high-scoring hits to sequence Y 26 SRI International Bioinformatics

27 Bayes Classifier P(protein has function X E-value, avg. rank, aln. length, etc.) best E-value protein has function X pwy directon avg. rank in BLAST output Number of queries % of query aligned adjacent rxns 27 SRI International Bioinformatics

28 Pathway Hole Filler Why should hole filler find things beyond the original genome annotation? Reverse BLAST searches more sensitive Reverse BLAST searches find second domains Integration of multiple evidence types 28 SRI International Bioinformatics

29 Caulobacter crescentus Pathway Holes 130 pathways containing 582 reactions 92 pathways contain 236 pathway holes Caulobacter holes filled: 77 holes filled at P >0.9 Previous functions of candidate hole fillers: No predicted function Correctly assigned single function Incorrectly assigned function Imprecise functional assignment BMC Bioinformatics 5: SRI International Bioinformatics

30 PathoLogic Step 5: Transport Inference Parser Problem: Write a program to query a genome annotation to compute the substrates an organism can transport Typical genome annotations for transporters: ATP transporter for ribose ribose ABC transporter D-ribose ATP transporter ABC transporter, membrane spanning protein [ribose] ABC transporter, membrane spanning protein [D-ribose] 30 SRI International Bioinformatics

31 Transport Inference Parser Input: ATP transporter of phosphonate Output: Structured description of transport activity Locates most transporters in genome annotation using keyword analysis Parse product name using a series of rules to identify: Transported substrate, co-substrate Influx/efflux Energy coupling mechanism Creates transport reaction object: phosphonate [periplasm] + H 2 O + ATP = phosphonate + P i + ADP 31 SRI International Bioinformatics

32 Transport Inference Parser Permits symbolic computation with transport activities: Compute transportable substrates of the cell Compute connectivity among compartments for substrates Facilitate reasoning about transport/metabolism connections Draw transport cartoon in protein pages, cellular overview 32 SRI International Bioinformatics

33 Tools 33 SRI International Bioinformatics

34 Pathway Tools Overviews and Omics Viewers Provide genome-scale visualizations of cellular networks Harness human visual system to interpret patterns in biological contexts Designed to avoid the hairball effect Generated automatically from PGDB Magnify, interrogate Omics viewers paint omics data onto overview diagrams Different perspectives on same dataset Use animation for multiple time points or conditions Paint any data that associates numbers with genes, proteins, reactions, or metabolites 34 SRI International Bioinformatics

35 35 SRI International Bioinformatics

36 Infer Anti-Microbial Drug Targets Infer drug targets as genes coding for enzymes that encode chokepoint reactions Two types of chokepoint reactions: Chokepoint analysis of Plasmodium falciparum: 216/303 reactions are chokepoints (73%) All 3 clinically proven anti-malarial drugs target chokepoints 21/24 biologically validated drug targets are chokepoints 11.2% of chokepoints are drug targets 3.4% of non-chokepoints are drug targets => Chokepoints are significantly enriched for drug targets Genome Research 14: SRI International Bioinformatics

37 Reachability Analysis of Metabolic Network Given: A PGDB for an organism A set of initial metabolites Infer: What set of products can be synthesized by the smallmolecule metabolism of the organism Can known growth medium yield known essential compounds? Romero and Karp, Pacific Symposium on Biocomputing, SRI International Bioinformatics

38 Algorithm: Forward Propagation Through Production System Each reaction becomes a production rule Each metabolite in nutrient set becomes an axiom Nutrient pool Products Transport Metabolite pool PGDB reaction set Fire reactions Reactants 38 SRI International Bioinformatics

39 Nutrients: A, B, C, E, F A + B W C + D X E + F Y W + Y Z Produced Compounds: W, Y, Z 39 SRI International Bioinformatics

40 Initial Metabolite Nutrient Set (Total: 21 compounds) Nutrients (8) (M61 Minimal growth medium) Nutrients (10) (Environment) Bootstrap Compounds (3) H +, Fe 2+, Mg 2+, K +, NH 3, SO 4 2-, PO 4 2-, Glucose Water, Oxygen, Trace elements (Mn 2+, Co 2+, Mo 2+, Ca 2+, Zn 2+, Cd 2+, Ni 2+, Cu 2+ ) ATP, NADP, CoA 40 SRI International Bioinformatics

41 Essential Compounds E. coli Total: 41 compounds Proteins (20) Amino acids Nucleic acids (DNA & RNA) (8) Nucleosides Cell membrane (3) Phospholipids Cell wall (10) Peptidoglycan precursors Outer cell wall precursors (Lipid-A, oligosaccharides) 41 SRI International Bioinformatics

42 Results Phase I: Forward propagation 21 initial compounds yielded only half of the 41 essential compounds for E. coli Phase II: Manually identify Bugs in EcoCyc (e.g., two objects for tryptophan) A B B C Incomplete knowledge of E. coli metabolic network A + B C + D Bootstrap compounds Missing initial protein substrates (e.g., ACP) Protein synthesis not represented Phase III: Forward propagation with 11 more initial metabolites Yielded all 41 essential compounds 42 SRI International Bioinformatics

43 Summary Pathway/Genome Databases MetaCyc non-redundant DB of literature-derived pathways 370 organism-specific PGDBs available through SRI at BioCyc.org Computational theories of biochemical machinery Pathway Tools software Extract pathways from genomes Omics data analysis Query, visualization, WWW publishing 43 SRI International Bioinformatics

44 BioCyc and Pathway Tools Availability BioCyc.org Web site and database files freely available to all Pathway Tools freely available to non-profits Macintosh, PC/Windows, PC/Linux 44 SRI International Bioinformatics

45 Acknowledgements SRI Suzanne Paley, Ron Caspi, Ingrid Keseler, Carol Fulcher, Markus Krummenacker, Alex Shearer, Tomer Altman, Joe Dale, Fred Gilham, Pallavi Kaipa EcoCyc Collaborators Julio Collado-Vides, Robert Gunsalus, Ian Paulsen MetaCyc Collaborators Sue Rhee, Peifen Zhang, Kate Dreher Lukas Mueller, Anuradha Pujar Funding sources: NIH National Center for Research Resources NIH National Institute of General Medical Sciences NIH National Human Genome Research Institute BioCyc.org Learn more from BioCyc webinars: biocyc.org/webinar.shtml 45 SRI International Bioinformatics