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PathoLogic Pathway Predictor

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PathoLogic Pathway Predictor. Inference of Metabolic Pathways. Gene Products. Genes/ORFs. DNA Sequences. Pathways. Reactions. Compounds. Annotated Genomic Sequence. Pathway/Genome Database. Pathways. Reactions. PathoLogic Software - PowerPoint PPT Presentation

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Page 1: PathoLogic Pathway Predictor

PathoLogic Pathway Predictor

Page 2: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsInference of Metabolic Pathways

Pathway/GenomeDatabase

Annotated GenomicSequence

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

Page 3: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsPathoLogic Functionality

Initialize schema for new PGDBTransform existing genome to PGDB formInfer metabolic pathways and store in PGDBInfer operons and store in PGDBAssemble Overview diagramAssist user with manual tasks

Assign enzymes to reactions they catalyze Identify false-positive pathway predictions Build protein complexes from monomers Infer transport reactions

Page 4: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsPathoLogic Input/Output

Inputs: File listing genetic elements

http://bioinformatics.ai.sri.com/ptools/genetic-elements.dat Files containing DNA sequence for each genetic element Files containing annotation for each genetic element MetaCyc database

Output: Pathway/genome database for the subject organism Reports that summarize:

Evidence contained in the input genome for the presence of reference pathways

Reactions missing from inferred pathways

Page 5: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsPathoLogic Analysis Phases

Trial parsing of input data files [few days] Initialize schema of new PGDB [3 min] Create DB objects for replicons, genes, proteins [5 min] Assign enzymes to reactions they catalyze

ferrochelatase [10 min / 1 week] glutamate 1-semialdehyde 2,1-aminomutase porphobilinogen deaminase

A C GB D E F

E1 E2

Page 6: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsPathoLogic Analysis Phases

From assigned reactions, infer what pathways are present [5 min / few days]

Define metabolic overview diagram [30 min]

Define protein complexes [few days]

Page 7: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsgenetic-elements.dat

ID TEST-CHROM-1NAME Chromosome 1TYPE :CHRSMCIRCULAR? NANNOT-FILE chrom1.pfSEQ-FILE chrom1.fsa//ID TEST-CHROM-2NAME Chromosome 2CIRCULAR? NANNOT-FILE /mydata/chrom2.gbkSEQ-FILE /mydata/chrom2.fna//

Page 8: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsFile Naming Conventions

One pair of sequence and annotation files for each genetic element

Sequence files: FASTA format suffix fsa or fna

Annotation file: Genbank format: suffix .gbk PathoLogic format: suffix .pf

Page 9: PathoLogic Pathway Predictor

SRI InternationalBioinformatics

Typical Problems Using Genbank Files With PathoLogic

Wrong qualifier names used: read PathoLogic documentation!

Extraneous information in a given qualifier

Check results of trial parse carefully

Page 10: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsGenBank File Format

Accepted feature types: CDS, tRNA, rRNA, misc_RNA

Accepted qualifiers: /locus_tag Unique ID [recm] /gene Gene name [req] /product [req] /EC_number [recm] /product_comment [opt] /gene_comment [opt] /alt_name Synonyms [opt] /pseudo Gene is a pseudogene [opt]

For multifunctional proteins, put each function in a separate /product line

Page 11: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsPathoLogic File Format

Each record starts with line containing an ID attribute Tab delimited Each record ends with a line containing //

One attribute-value pair is allowed per line Use multiple FUNCTION lines for multifunctional proteins

Lines starting with ‘;’ are comment lines

Valid attributes are: ID, NAME, SYNONYM STARTBASE, ENDBASE, GENE-COMMENT FUNCTION, PRODUCT-TYPE, EC, FUNCTION-COMMENT DBLINK INTRON

Page 12: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsPathoLogic File Format

ID TP0734NAME deoDSTARTBASE 799084ENDBASE 799785FUNCTION purine nucleoside phosphorylaseDBLINK PID:g3323039PRODUCT-TYPE PGENE-COMMENT similar to GP:1638807 percent identity: 57.51;

identified by sequence similarity; putative//ID TP0735NAME gltASTARTBASE 799867ENDBASE 801423FUNCTION glutamate synthaseDBLINK PID:g3323040PRODUCT-TYPE P

Page 13: PathoLogic Pathway Predictor

SRI InternationalBioinformatics

Before you start: What to do when an error occursMost Navigator errors are automatically trapped –

debugging information is saved to error.tmp file.All other errors (including most PathoLogic

errors) will cause software to drop into the Lisp debugger

Unix: error message will show up in the original terminal window from which you started Pathway Tools.

Windows: Error message will show up in the Lisp console. The Lisp console usually starts out iconified – its icon is a blue bust of Franz Liszt

2 goals when an error occurs: Try to continue working Obtain enough information for a bug report to send to

pathway-tools support team.

Page 14: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsThe Lisp Debugger

Sample error (details and number of restart actions differ for each case)Error: Received signal number 2 (Keyboard interrupt)

Restart actions (select using :continue):

0: continue computation

1: Return to command level

2: Pathway Tools version 10.0 top level

3: Exit Pathway Tools version 10.0

[1c] EC(2):

To generate debugging information (stack backtrace)::zoom :count :all

To continue from error, find a restart that takes you to the top level – in this case, number 2:cont 2

To exit Pathway Tools::exit

Page 15: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsHow to report an error

Determine if problem is reproducible, and how to reproduce it (make sure you have all the latest patches installed)

Send email to [email protected] containing:

Pathway Tools version number and platform Description of exactly what you were doing (which command

you invoked, what you typed, etc.) or instructions for how to reproduce the problem

error.tmp file, if one was generated If software breaks into the lisp debugger, the complete error

message and stack backtrace (obtained using the command :zoom :count :all, as described on previous slide)

Page 16: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsUsing the PPP GUI to Create a

Pathway/Genome Database

Input Project Information Organism -> Create New

Page 17: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsInput Project Information

Page 18: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsNext Steps

Trial Parse Build -> Trial Parse Fix any errors in input files

Build pathway/genome database Build -> Automated Build

Page 19: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsPathoLogic Parser Output

Page 20: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsAssign Enzymes to

Reactions

MatchMatch

Gene Gene productproduct

5.1.3.2

UDP-glucose-4-epimerase

yesyes

AssignAssign

nono

Probable enzymeProbable enzyme-ase-ase

nono yesyes

Not a metabolic Not a metabolic enzymeenzyme

Manually Manually searchsearch

yesyes

AssignAssign

nono

Can’t AssignCan’t Assign

MetaCyc

UDP-D-glucose UDP-galactose

Page 21: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsEnzyme Name Matcher

Matches on full enzyme nameMatch is case-insensitive and removes the

punctuation characters “ -_(){}',:”Also matches after removal of prefixes and

suffixes such as: “Putative”, “Hypothetical”, etc alpha|beta|…|catalytic|inducible chain|subunit|component Parenthetical gene name

Page 22: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsEnzyme Name Matcher

For names that do not match, software identifies probable metabolic enzymes as those

Containing “ase” Not containing keywords such as

“sensor kinase” “topoisomerase” “protein kinase” “peptidase” Etc

Research unknown enzymes MetaCyc, Swiss-Prot, PubMed

Page 23: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsEnzyme Name to Reaction

Mapping

See also file PTools Tutorial/PathoLogic Reports/name-matching-report.txt

Page 24: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsManual Polishing

Refine -> Assign Probable Enzymes Do this first

Refine -> Rescore Pathways Redo after assigning enzymes

Refine -> Create Protein Complexes Can be done at any time

Refine -> Assign Modified Proteins Can be done at any time

Refine -> Transport Identification Parser Can be done at any time

Refine -> Pathway Hole Filler

Refine -> Predict Transcription Units

Refine -> Update Overview Do this last, and repeat after any material changes to PGDB

Page 25: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsAssign Probable Enzymes

Page 26: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsHow to find reactions for

probable enzymes

First, verify that enzyme name describes a specific, metabolic function

Search for fragment of name in MetaCyc – you may be able to find a match that PathoLogic missed

Look up protein in SwissProt or other DBsSearch for gene name in PGDB for related

organism (bear in mind that gene names are not reliable indicators of function, so check carefully)

Search for function name in PubMedOther…

Page 27: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsManual Polishing

Refine -> Assign Probable Enzymes

Refine -> Rescore Pathways

Refine -> Create Protein Complexes

Refine -> Assign Modified Proteins

Refine -> Transport Identification Parser

Refine -> Pathway Hole Filler

Refine -> Predict Transcription Units

Refine -> Run Consistency Checker

Refine -> Update Overview

Page 28: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsAutomated Pathway Inference

All pathways in MetaCyc for which there is at least one enzyme identified in the target organism are considered for possible inclusion.

Algorithm errs on side of inclusivity – easier to manually delete a pathway from an organism than to find a pathway that should have been predicted but wasn’t.

Page 29: PathoLogic Pathway Predictor

SRI InternationalBioinformatics

Considerations taken into account when deciding whether or not a pathway should be inferred: Is there a unique enzyme – an enzyme not involved in any

other pathway? Does the organism fall in the expected taxonomic domain of

the pathway? Is this pathway part of a variant set, and, if so, is there more

evidence for some other variant? If there is no unique enzyme:

Is there evidence for more than one enzyme? If a biosynthetic pathway, is there evidence for final reaction(s)? If a degradation pathway, is there evidence for initial reaction(s)? If an energy metabolism pathway, is there evidence for more than half the

reactions?

Page 30: PathoLogic Pathway Predictor

SRI InternationalBioinformatics

Assigning Evidence Scores to Predicted Pathways

X|Y|Z denotes score for P in O where:

X = total number of reactions in P Y = enzymes catalyzing number of reactions for which there is

evidence in O Z = number of Y reactions that are used in other pathways in O

Page 31: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsManual Pruning of Pathways

Use pathway evidence report Coloring scheme aids in assessing pathway evidence

Phase I: Prune extra variant pathways

Rescore pathways, re-generate pathway evidence report

Phase II: Prune pathways unlikely to be present No/few unique enzymes Most pathway steps present because they are used in another pathway Pathway very unlikely to be present in this organism Nonspecific enzyme name assigned to a pathway step

Page 32: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsCaveats

Cannot predict pathways not present in MetaCyc

Evidence for short pathways is hard to interpret

Since many reactions occur in multiple pathways, some false positives

Page 33: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsOutput from PPP

Pathway/genome database

Summary pages Pathway evidence page

Click “Summary of Organisms”, then click organism name, then click “Pathway Evidence”, then click “Save Pathway Report”

Missing enzymes report

Directory tree containing sequence files, reports, etc.

Page 34: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsResulting Directory Structure

ROOT/ptools-local/pgdbs/user/ORGIDcyc/VERSION/ input

organism.dat organism-init.dat genetic-elements.dat annotation files sequence files

reports name-matching-report.txt trial-parse-report.txt

kb ORGIDbase.ocelot

data overview.graph

released -> VERSION

Page 35: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsManual Polishing

Refine -> Assign Probable Enzymes

Refine -> Rescore Pathways

Refine -> Create Protein Complexes

Refine -> Assign Modified Proteins

Refine -> Transport Identification Parser

Refine -> Pathway Hole Filler

Refine -> Predict Transcription Units

Refine -> Run Consistency Checker

Refine -> Update Overview

Page 36: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsCreating Protein Complexes

Page 37: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsComplex Subunits

Stoichiometries

Page 38: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsManual Polishing

Refine -> Assign Probable Enzymes

Refine -> Re-run Name Matcher

Refine -> Create Protein Complexes

Refine -> Assign Modified Proteins

Refine -> Transport Identification Parser

Refine -> Pathway Hole Filler

Refine -> Predict Transcription Units

Refine -> Run Consistency Checker

Refine -> Update Overview

Page 39: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsProteins as Reaction Substrates

Page 40: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsManual polishing

Refine -> Assign Probable Enzymes

Refine -> Rescore Pathways

Refine -> Create Protein Complexes

Refine -> Assign Modified Proteins

Refine -> Transport Identification Parser

Refine -> Pathway Hole Filler

Refine -> Predict Transcription Units

Refine -> Run Consistency Checker

Refine -> Update Overview

michelle
probably need to expand the description of the algorithm for the operon predictor (specifically, the use of log likelihoods and why intergenic distance and the fact that no nucleotide-based features, other than distance, are used)
Page 41: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsNomenclature

•WO pair = pair of genes within an operon

•TUB pair = pair of genes at a transcription unit boundary (delineate operons)

Page 42: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsOperation of the operon

predictor

For each contiguous gene pair, predict whether gene pairs are within the same operon or at a transcription unit boundary

Use pairwise predictions to identify potential operons

AB = TUB pair

BC = WO pair operon = BCD

CD = WO pair

DE = TUB pair

A B C D E

Page 43: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsOperon predictor

Predicts operon gene pairs based on: intergenic distance between genes genes in the same functional class

Typically used for operon prediction We use method from Salgado et al, PNAS (2000) as a

starting point. Uses E. coli experimentally verified data as a training set. Compute log likelihood of two genes being WO or TUB pair based

on intergenic distance.

Page 44: PathoLogic Pathway Predictor

SRI InternationalBioinformaticsOperon predictor

Additional features easily computed from a PGDB

1. both genes products enzymes in the same metabolic pathway

2. both gene products monomers in the same protein complex3. one gene product transports a substrate for a metabolic

pathway in which the other gene product is involved as an enzyme

4. a gene upstream or downstream from the gene pair (and within the same directon) is related to either one of the genes in the pair as per features 1, 2 and 3 above.