Pathway Bioinformatics: Inference, Visualization, and Analysis · 2008-04-21 · 2 SRI International Bioinformatics Overview zMotivations zPathway/genome databases zBioCyc collection

1 SRI International Bioinformatics

Pathway Bioinformatics: Inference, Visualization, and Analysis

Peter D. Karp, Ph.D.Bioinformatics Research Group

SRI [email protected]

BioCyc.orgEcoCyc.org, MetaCyc.org, HumanCyc.org


Overview

MotivationsPathway/genome databases

BioCyc collectionEcoCyc, MetaCyc

Pathway Tools softwareVisualization, Editing, AnalysisInference tools: Pathway hole filler, Transport inference parserReachability analysis of metabolic network

ApplicationsNew levels of genome annotationOmics data analysisPublish curated pathway/genome database on the web


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 organismIntegrate literature-based information with computational predictions

Curated by experts for that organismNo one group can curate all the world’s genomesDistribute workload across a community of experts to create a community resource


Rationale for MODs

Each “complete” genome is incomplete in several respects:40%-60% of genes have no assigned functionRoughly 7% of those assigned functions are incorrectMany 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 organismInterpret omics data in a pathway contextIn silico prediction of essential genesCharacterize systems properties of metabolic and genetic networks


BioCyc Collection of Pathway/Genome Databases

Pathway/Genome Database (PGDB) –combines information about

Pathways, reactions, substratesEnzymes, transportersGenes, repliconsTranscription factors/sites, promoters, operons

Tier 1: Literature-Derived PGDBsMetaCycEcoCyc -- Escherichia coli K-12

Tier 2: Computationally-derived DBs, Some Curation -- 20 PGDBs

HumanCycMycobacterium tuberculosis

Tier 3: Computationally-derived DBs, No Curation -- 349 DBs


Pathway Tools Overview

Pathway/GenomeEditors

Pathway/GenomeDatabase

PathoLogicAnnotatedGenome

MetaCycReference

Pathway DB

Pathway/GenomeNavigator


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 operonsPredicts metabolic networkPredicts which genes code for missing enzymes in metabolic pathways Infers transport reactions from transporter names

Bioinformatics 18:S225 2002


Pathway Tools Software:Pathway/Genome Editors

Interactively update PGDBs with graphical editors

Support geographically distributed teams of curators with object database system

Gene editorProtein editorReaction editorCompound editorPathway editorOperon editorPublication editor


Pathway Tools Software:Pathway/Genome Navigator

Querying and visualization of:PathwaysReactionsMetabolitesProteinsGenesChromosomes

Two modes of operation:Web modeDesktop modeMost functionality shared, but each has unique functionality


Pathway Tools Ontology / Schema

Ontology classes: 1621Many datatypes from genomes to pathwaysClassification schemes for pathways, chemical compounds, enzymatic reactions (EC system)Cell Component Ontology Protein Feature ontology

Comprehensive set of 221 attributes and relationshipsEvidence codes, supporting citations


BioCyc Tier 3

Tier 3 DBs created by subjecting annotated genomes to PathoLogic computational processing pipeline:

Pathway predictionPathway Hole FillingOperon prediction (bacteria)Transport Inference Parser

349 PGDBsSource: CMR database


Pathway Tools Software: PGDBs Created Outside SRI

1,300+ licensees: 75+ groups applying software to 200+ organisms

Saccharomyces cerevisiae, SGD project, Stanford UniversityMouse, MGD, Jackson LaboratorydictyBase, Northwestern UniversityUnder development:

CGD (Candida albicans), Stanford UniversityDrosophila, P. Ebert in collaboration with FlyBaseC. elegans, P. Ebert in collaboration with WormBase

Planned:RGD (Rat), Medical College of Wisconsin

Arabidopsis thaliana, TAIR, Carnegie Institution of WashingtonPlantCyc, ~20 plant PGDBs, Carnegie Institution of WashingtonSix Solanaceae species, Cornell University GrameneDB, Cold Spring Harbor LaboratoryMedicago truncatula, Samuel Roberts Noble Foundation


EcoCyc Project – EcoCyc.orgE. coli Encyclopedia

Review-level Model-Organism Database for E. coliTracks evolving annotation of the E. coli genome and cellular networksThe two paradigms of EcoCyc

“Multi-dimensional annotation of the E. coli K-12 genome”Positions of genes; functions of gene products – 76% / 66% expGene Ontology terms; MultiFun termsGene product summaries and literature citationsEvidence codesMultimeric complexesMetabolic pathwaysRegulation of transcription initiation

Nuc. Acids Res. 35:7577 2007 ASM News 70:25 2004 Science 293:2040

Karp, Gunsalus, Collado-Vides, Paulsen


EcoCyc = E.coli Dataset + Pathway/Genome Navigator

Genes: 4,471

Proteins: 4,464Complexes: 808

RNAs: 285

Reactions:Metabolic: 923Transport: 259

Pathways: 224

Compounds: 1,227

URL: EcoCyc.org

Gene Regulation:Operons: 3,187Trans Factors: 179Promoters: 1,343

TF Binding Sites: 1,189

Citations: 16,153


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


Paradigm 2: EcoCyc as Computational Symbolic Theory

Highly structured, high-fidelity knowledge representation provides computable informationEach molecular species defined as a DB object

Genes, proteins, small moleculesEach molecular interaction defined as a DB object

Metabolic reactionsTransport reactionsTranscriptional regulation of gene expression

220 database fields capture extensive properties and relationships


EcoCyc Procedures

DB updates performed by 5 staff curatorsInformation gathered from biomedical literature

Enter data into structured database fieldsAuthor extensive summariesUpdate evidence codes

Corrections submitted by E. coli researchers

Four releases per year

Quality assurance of data and softwareEvaluate database consistency constraintsPerform element balancing of reactionsRun other checking programs


EcoCyc Accelerates Science

ExperimentalistsE. coli experimentalistsExperimentalists working with other microbesAnalysis of expression data

Computational biologists Biological research using computational methodsGenome annotationStudy connectivity of E. coli metabolic networkStudy phylogentic extent of metabolic pathways and enzymes in all domains of life

BioinformaticistsTraining 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


MetaCyc: Metabolic Encyclopedia

Describe a representative sample of every experimentally determined metabolic pathwayDescribe properties of metabolic enzymes

Literature-based DB with extensive references and commentaryPathways, reactions, enzymes, substrates

Jointly developed by P. Karp, R. Caspi, C. Fulcher, SRI InternationalL. Mueller, A. Pujar, Cornell UnivS. Rhee, P. Zhang, Carnegie Institution

Nucleic Acids Research 2008


Applications of MetaCyc

Reference source on metabolic pathways

Metabolic engineeringFind enzymes with desired activities, regulatory propertiesDetermine cofactor requirements

Predict pathways from genomes

Systematic studies of metabolism

Computer-aided education


MetaCyc Data -- Version 12.0

16,335Citations

1,108Organisms

6,719Small Molecules

4,731Enzymes

6,739Reactions

1036Pathways


Taxonomic Distribution ofMetaCyc Pathways – version 12.0

86Archaea

154Fungi

119Mammals

452Green Plants

691Bacteria


Family of Pathway/GenomeDatabases

MetaCyc

EcoCycCauloCycAraCyc

MtbRvCycHumanCyc


PathoLogic Step 1: Translate Genome to PGDBPathway/Genome

DatabaseAnnotated Genomic

Sequence

Genes/ORFs

Gene Products

DNA Sequences

Reactions

Pathways

Compounds

Multi-organism PathwayDatabase (MetaCyc)

PathoLogic Software

Integrates genome and pathway data to identify

putative metabolic networks

Genomic Map

Genes

Gene Products

Reactions

Pathways

Compounds


PathoLogic Step 4: Pathway Hole Filler

Definition: Pathway Holes are reactions in metabolic pathways for which no enzyme is identified

holesholes

LL--aspartateaspartate iminoaspartateiminoaspartate

quinolinatequinolinate

nicotinate nicotinate nucleotidenucleotide

deamidodeamido--NADNAD

NADNAD

1.4.31.4.3..--

2.7.7.182.7.7.18

quinolinate synthetasequinolinate synthetasenadAnadA

n.n. pyrophosphorylasen.n. pyrophosphorylasenadCnadC

NAD+ synthetase, NH3 NAD+ synthetase, NH3 --dependentdependent

CC3619CC3619

6.3.5.16.3.5.1


organism 1 enzyme A

organism 2 enzyme A

organism 5 enzyme A

organism 4 enzyme A

organism 3 enzyme A

organism 8 enzyme A

organism 7 enzyme A

organism 6 enzyme A

Step 1: Query UniProt for all sequences having

EC# of pathway hole

Step 2: BLAST against target

genome Step 3 & 4: Consolidate

hits and evaluate evidence

7 queries have high-scoring hits to sequence Y

gene Ygene X

gene Z


Bayes Classifier

P(protein has function X|P(protein has function X|EE--value, avg. rank, aln. length, etc.)value, avg. rank, aln. length, etc.)

protein has protein has function Xfunction X

% of query % of query alignedaligned

avg. rank in avg. rank in BLAST outputBLAST output

bestbestEE--valuevalue

Number of Number of queriesqueries

pwy pwy directondirecton

adjacent adjacent rxnsrxns


Pathway Hole Filler

Why should hole filler find things beyond the original genome annotation?

Reverse BLAST searches more sensitiveReverse BLAST searches find second domainsIntegration of multiple evidence types


Caulobacter crescentus Pathway Holes

130 pathways containing 582 reactions92 pathways contain 236 pathway holes

Caulobacter holes filled:77 holes filled at P >0.9

Previous functions of candidate hole fillers:No predicted functionCorrectly assigned single functionIncorrectly assigned functionImprecise functional assignment

BMC Bioinformatics 5:76 2004


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 riboseribose ABC transporterD-ribose ATP transporterABC transporter, membrane spanning protein [ribose]ABC transporter, membrane spanning protein [D-ribose]


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-substrateInflux/effluxEnergy coupling mechanism

Creates transport reaction object:

phosphonate[periplasm] + H2O + ATP = phosphonate + Pi + ADP


Transport Inference Parser

Permits symbolic computation with transport activities:Compute transportable substrates of the cellCompute connectivity among compartments for substratesFacilitate reasoning about transport/metabolism connectionsDraw transport cartoon in protein pages, cellular overview


Tools


Pathway Tools Overviews and Omics Viewers

Designed to avoid the hairball effectGenerated automatically from PGDBMagnify, interrogateOmics viewers paint omics data onto

overview diagramsDifferent perspectives on same datasetUse animation for multiple time points or conditionsPaint any data that associates numbers with genes, proteins, reactions, or metabolites

Provide genome-scale visualizations of cellular networksHarness human visual system to interpret patterns in biological

contexts



Infer Anti-Microbial Drug TargetsInfer 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 chokepoints21/24 biologically validated drug targets are chokepoints11.2% of chokepoints are drug targets3.4% of non-chokepoints are drug targets=> Chokepoints are significantly enriched for drug targets

Genome Research 14:917 2004


Reachability Analysis of Metabolic Network

Given:A PGDB for an organismA set of initial metabolites

Infer:What set of products can be synthesized by the small-molecule metabolism of the organism

Can known growth medium yield known essential compounds?

Romero and Karp, Pacific Symposium on Biocomputing, 2001


Algorithm: Forward PropagationThrough Production System

Each reaction becomes a production ruleEach metabolite in nutrient set becomes an axiom

Nutrientpool

Metabolitepool

“Fire”reactions

Transport

Products

Reactants

PGDBreaction

set


A + B W

E + F Y

C + D X

W + Y Z

Nutrients: A, B, C, E, F

Produced Compounds: W, Y, Z


Initial Metabolite Nutrient Set (Total: 21 compounds)

Nutrients (8) (M61 Minimal growth medium)

H+, Fe2+, Mg2+, K+, NH3, SO4

2-, PO42-, Glucose

Nutrients (10) (Environment)

Water, Oxygen, Trace elements (Mn2+, Co2+, Mo2+, Ca2+, Zn2+, Cd2+, Ni2+, Cu2+)

Bootstrap Compounds (3) ATP, NADP, CoA


Essential CompoundsE. coli Total: 41 compounds

Proteins (20)Amino acids

Nucleic acids (DNA & RNA) (8)Nucleosides

Cell membrane (3)Phospholipids

Cell wall (10)Peptidoglycan precursorsOuter cell wall precursors (Lipid-A, oligosaccharides)


Results

Phase I: Forward propagation21 initial compounds yielded only half of the 41 essential compounds for E. coli

Phase II: Manually identifyBugs in EcoCyc (e.g., two objects for tryptophan)

A B B’ CIncomplete 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


Summary

Pathway/Genome DatabasesMetaCyc non-redundant DB of literature-derived pathways370 organism-specific PGDBs available through SRI at BioCyc.orgComputational theories of biochemical machinery

Pathway Tools softwareExtract pathways from genomesOmics data analysisQuery, visualization, WWW publishing


BioCyc and Pathway Tools Availability

BioCyc.org Web site and database files freely available to all

Pathway Tools freely available to non-profitsMacintosh, PC/Windows, PC/Linux


Acknowledgements

SRISuzanne Paley, Ron Caspi, Ingrid Keseler, Carol Fulcher, Markus Krummenacker, Alex Shearer, Tomer Altman, Joe Dale, Fred Gilham, Pallavi Kaipa

EcoCyc CollaboratorsJulio Collado-Vides, Robert Gunsalus, Ian Paulsen

MetaCyc CollaboratorsSue Rhee, Peifen Zhang, Kate DreherLukas Mueller, Anuradha Pujar

Funding sources:NIH National Center for Research ResourcesNIH National Institute of General Medical SciencesNIH National Human Genome Research Institute

BioCyc.org

Learn more from BioCyc webinars: biocyc.org/webinar.shtml

Documents

Pathway Bioinformatics: Inference, Visualization, and Analysis · 2008-04-21 · 2 SRI International Bioinformatics Overview zMotivations zPathway/genome databases zBioCyc collection