PathoLogic Pathway Predictor
Dec 24, 2015
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
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 Fill pathway holes
SRI InternationalBioinformaticsPathoLogic Input/Output
Inputs: List of all genetic elements
Enter using GUI or provide a file Files containing annotation for each genetic element Files containing DNA sequence for each genetic element MetaCyc database
Output: Pathway/genome database for the subject organism Reports that summarize:
Evidence in the input genome for the presence of reference pathways Reactions missing from inferred pathways
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
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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
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] /db_xref DB:AccessionID [opt] /go_component, /go_function, /go_process GO terms [opt]
For multifunctional proteins, put each function in a separate /product line
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 GO INTRON
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 PGO glutamate synthase (NADPH) activity [goid 0004355]
[evidence IDA] [pmid 4565085]
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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.
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
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)
SRI InternationalBioinformaticsUsing the PPP GUI to Create a
Pathway/Genome Database
Input Project Information Organism -> Create New Creates directory structure for new PGDB Creates and saves empty PGDB, populated only with objects
common to all PGDBs (schema classes, elements, etc.) and data you entered in the form.
Offers to invoke Replicon Editor
SRI InternationalBioinformaticsInput Project Information
SRI InternationalBioinformaticsEnter Replicon Information
For each replicon Name Type: chromosome, plasmid, etc. Circular? Annotation file Sequence file (optional) Contigs (optional) Links to other DBs (optional)
GUI-Based entry Build->Specify Replicons
File-Based Entry Create genetic-elements.dat file using template provided
SRI InternationalBioinformatics GUI-Based Replicon Entry
SRI InternationalBioinformaticsBatch Entry of Replicon Info
File /<orgid>cyc/<version>/input/genetic-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//
SRI InternationalBioinformaticsSpecify Reference PGDB(s)
This step is optional, and most users will omit itMetaCyc is always the primary reference PGDBSpecify additional reference PGDB if you have
your own curated PGDB which has: Pathways and/or reactions that are not in MetaCyc Manual functional assignments, with names similar to current
genomeThere is no point specifying any of our PGDBs as
references, only your own curated PGDBs.
SRI InternationalBioinformaticsBuilding the PGDB
Trial Parse Build -> Trial Parse Check output to ensure numbers “look right”
Same number of gene start positions, end positions, names Did my file contain EC numbers? Were they detected? Did my file contain RNAs? Were they detected?
Fix any errors in input filesBuild pathway/genome database
Build -> Automated Build
SRI InternationalBioinformaticsPathoLogic Parser Output
SRI InternationalBioinformaticsAutomated Build
Parses input filesCreates objects for every gene and gene productUses EC numbers, GO annotations and name
matcher to match enzymes to reactions in MetaCyc
Imports catalyzed enzymes and compounds from MetaCyc
Generates list of likely enzymes that couldn’t be assigned
Infers pathways likely to be presentGenerates Cellular Overview Diagram (first pass)Generates reports
SRI InternationalBioinformaticsMatching Enzymes to Reactions
Matches on full EC number (partial ECs ignored)Matches on Molecular Function GO terms
If definition of GO term includes cross-reference either to an EC number or to a MetaCyc reaction.
Matches on full enzyme name Match 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
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
User should research unknown enzymes MetaCyc, Swiss-Prot, PubMed
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Stored in ORGIDcyc/VERSION/reports/name-matching-report.txt
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.
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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?
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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
SRI InternationalBioinformaticsPathway Evidence Report
On Organism Summary Page in Navigator, button “Generate Pathway Evidence Report”
Report saved as HTML file, view in browserHierarchical listing of all inferred pathways
“Pathway Glyph” shows evidence graphically Steps with/without enzymes (green/black) Steps that are unique to pathway (orange) Steps filled by Pathway Hole Filler (blue)
Counts reactions in pathway, with evidence, in other pathways
Lists other pathways that share reactions Link to pathway in MetaCyc
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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
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
Next generation pathway inference algorithm is work currently in progress!
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.
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
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
SRI InternationalBioinformaticsAssign Probable Enzymes
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 UniProt 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…