The! methods! used! to! add! annotations! to! the! M.# tuberculosis! H37Rv! genome! and! compare!the!organisms!within!the!phylogenetic!profile!provide!a!number!of!interesting! results.! However,! some! of! these! methods! also! cause! potential! confounding! factors! in! terms!of!the!interpretation!of!results.!
One!of!the!first!potential!factors!affecting!this!work!is!the!choice!of!KEGG!as!the!main! pathway!database!(Ogata!et!al.,!1999).!The!choice!of!pathway!database!can!have!a!huge! effect!on!the!results!for!a!number!of!reasons.!Firstly,!pathway!description!and!naming! varies!widely!across!databases!and!literature.!Many!pathways!overlap!and!this!causes! discrepancies! as! well! as! repetition! within! databases.! Additionally,! some! reactions! marked! as! missing! may! in! fact! only! be! artefacts! of! other! pathways.! For! example,! if! reactions! are! replicated! within! other! pathways,! they! may! show! up! in! a! pathway! or! a! branch!of!a!pathway,!even!if!that!section!of!a!pathway!doesn’t!exist!within!the!organism.! This! may! also! cause! problems! in! cases! where! compounds! or! metabolites! are! used! in! other!pathways.!For!example,!reactions!may!produce!a!particular!metabolite!within!a! pathway,! with! all! following! reactions! showing! as! nonJannotated;! it! could! be! assumed! that! the! subsequent! reactions! might! be! missing! in! the! organism.! However,! this!
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compound!may!be!used!in!another!pathway,!and!might!not!prove!to!be!actually!missing.! Unlike! KEGG,! MetaCyc! unlinks! commonly! repeated! reactions,! thus! reducing! the! redundancy!in!the!database.!KEGG!pathway!maps!include!all!known!reactions!related!to! a! particular! topic! or! goal,! regardless! of! the! species! or! kingdoms! in! which! they! occur.! Since!many!pathway!variants!occur,!when!different!organisms!use!different!reactions! or! pathways! in! order! to! achieve! the! same! metabolic! goal,! many! reactions! in! KEGG! diagrams!are!specific!for!unrelated!organisms,!such!as!perhaps!mammals,!and!do!not! exist!in!Mtb!(Caspi!et!al.,!2013).!The!main!annotations!and!pathways!in!this!research! were! extracted! from! the! KEGG! database,! because! the! diagrams! in! KEGG! were! clearer! and!more!manageable.!Since!many!of!these!KEGG!diagrams!were!quite!large,!some!were! broken! into! pieces! of! pathways! derived! from! the! MetaCyc! database.! Since! functional! annotations! were! obtained! from! KEGG,! there! could! be! false! negatives! in! that! some! MetaCyc! reactions! are! not! included! in! the! KEGG! database.! Additionally,! and! probably! having!a!stronger!effect!on!the!results,!there!could!be!false!positives!for!characterised! and! missing! reactions! due! to! the! overlap! between! pathways.! Reactions! that! were! characterised!or!found!to!be!missing!could!rather!be!artefacts!of!other!pathways.!This! could! affect! certain! pathways! by! enabling! them! to! be! characterised! and! deemed! functional! in! M.# tuberculosis# H37Rv! when! in! fact! they! are! only! present! because! the! reactions! are! shared! with! other! pathways.! Thus! the! obtained! results! might! require! further!experimentation!in!order!to!confirm!or!refute!them.!
A! second! potential! issue! is! the! use! of! primary! sequence! structure! to! determine! homology! and! EC! number! to! determine! functional! characterisation.! Protein! matches! were!made!to!annotate!the!genome!of!M.#tuberculosis#H37Rv!by!using!BLASTP!to!find! protein! matches! for! already! annotated! enzymes! in! KEGG.! Studies! have! found! that! proteins!in!pathway!alignments!(aligning!pathways!from!two!or!more!organisms!to!find! proteins)!did!not!necessarily!pair!with!the!best!sequence!match!of!the!other!pathway.! This!suggests!that!if!a!multifunctional!protein!of!one!organism!experiences!duplication! and! specialisation! in! another! organism,! then! it! could! have! different! orthologs! in! different! metabolic! pathways! (Kelley! et! al.,! 2003).! This! could! have! an! effect! on! the! research!conducted!here,!since!additional!reactions!were!annotated!based!on!sequence! similarity!and!not!on!proteinJprotein!interactions.!In!addition!to!sequence!similarity,!EC! number! assignments! were! used! to! characterise! some! proteins.! A! previous! study!
aligning!the!metabolic!pathways!of!Escherichia#coli!and!Saccharomyces#cerevisiae!used! EC! number! rather! than! sequence! similarity! to! form! alignments;! this! allowed! the! authors! to! locate! functional! orthologs! resulting! from! both! convergent! evolution,! in! which!nonJhomologous!proteins!evolved!the!same!function,!or!divergent!evolution,!in! which! orthologous! proteins! diverge! in! sequence! while! retaining! the! same! function.! Thus!they!showed!the!efficacy!of!using!functional!classification!in!addition!to!sequenceJ based!classification!in!order!to!compare!pathways!(Pinter!et!al.,!2005).!In!the!current! study,!functional!classification!(for!example!EC!numbers!and!GO!terms)!has!been!used! in! addition! to! sequenceJsimilarity! in! order! to! complete! functional! gaps! in! the! metabolism!of!Mtb.!This!also!provides!a!basis!for!the!technique!of!using!a!protein’s!EC! number!to!assign!that!protein!to!a!pathway!or!reaction.!However,!this!means!that!these! functional!classifications!rely!on!the!accuracy!of!the!EC!numbers!and!GO!terms.! The!phylogenetic!comparison!itself!can!be!affected!by!the!use!of!the!KEGG!database.!It!is! hard!to!compare!KEGG!maps!phylogenetically!because!of!the!fact!that!they!encompass! all!reactions!related!to!a!particular!topic;!if!one!organism!has!less!coverage!of!a!pathway! it! could! be! because! of! different! branches! within! the! pathway.! Alternatively,! two! organisms! could! show! the! same! number! of! reactions! for! a! pathway,! but! the! actual! mapping! shows! entirely! different! metabolisms,! with! the! two! organisms! utilising! completely!different!portions!of!the!map!with!no!or!little!overlap!(Altman!et!al.,!2013).! While!this!means!that!pathway!comparisons!are!more!uncertain!and!less!predictive!in! KEGG,!the!larger!structure!of!KEGG!maps!makes!it!easier!to!compare!all!pathways!of!the! metabolome! as! a! whole.! In! this! study,! this! factor! should! not! be! a! major! problem! in! comparing!pathway!totals!of!organisms!to!M.#tuberculosis#H37Rv!since!these!totals!were! calculated!using!only!orthologous!proteins!of!M.#tuberculosis#H37Rv.!On!the!other!hand,! organisms!(besides!M.#tuberculosis#H37Rv)!shown!to!have!the!same!number!of!proteins! in!pathways!might!have!different!parts!of!the!pathways!and!thus!cannot!be!compared!to! one! another.! Additionally,! the! protein! counts! of! proteins! derived! from! the! KEGG! orthology! lists! include! duplicates! of! proteins;! when! a! protein! catalyses! multiple! reactions! within! one! pathway,! it! is! counted! twice! in! these! summary! totals.! This! will! sometimes!lead!to!the!KEGG!protein!counts!being!greater!than!those!summarised!from! the!matrix,!and!so!this!must!be!taken!into!account!when!evaluating!these!results.!
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The!prediction!of!missing!proteins!can!also!be!effected!by!the!used!of!BLASTP!to!find! protein! matches! with! proteins! from! closely! related! organisms! and! characterise! pathways!in!M.#tuberculosis!H37Rv.!In!previous!research,!wellJcharacterised!pathways! of! model! organisms! have! been! used! to! predict! homologous! pathways! in! other! prokaryotes!(Mao!et!al.,!2012).!Pathway!holes!are!missing!pieces!in!a!mapped!pathway,! and! can! result! from! two! conditions! when! performing! pathway! prediction! in! this! manner.!Firstly,!genes!of!the!template!pathways!may!not!always!map!to!any!gene!in!the! organism.!Secondly,!the!organism!may!possess!reactions!or!pathways!that!the!template! pathways!do!not!have.!Thus,!some!‘missing’!reactions!may!be!false!positives!while!other! reactions!not!classified!as!‘missing’!may!prove!to!be!false!negatives.!