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Comparative Interactomics
   with FunCoup 2.0

      Erik Sonnhammer

  Stockholm Bioinformatics Centre
     Science for Life Laboratory
  Dept. Biochemistry and Biophysics
        Stockholm University
How to map the human interactome?

• Genes: ~22000
• Interactions: 100000-300000?
• Known direct interactions:
  ~74000 (Intact)

• Experiments have high false
  negative and false positive rates.

• → Most interactions need
  to be inferred combinatorially
FunCoup:

                        Predicting
  Functional Coupling Between Genes/Proteins
        Using Genomics Data and Orthology



• Alexeyenko et al., NAR 40:D821 (2012)
• Alexeyenko & Sonnhammer, Genome Research 19:1107 (2009)
Co-expression
                         patterns
                                             Phylogenetic
                                             profiles
         Domain
         interactions




Shared
transcription
                        FunCoup                        Protein-protein
                                                       interactions
factor
binding




        Shared              Orthology                           Genetic
                                         Subcellular
        miRNA                                                   interactions
                                         co-localisation
        targeting




                          Other
                        Organisms
Naïve Bayesian training
                            -1.0                     1.0
Continuous variable
                                           0.6        1.0
Discrete categories

Extract links


                                   +   +         +
Test against positive and
”negative” reference
datasets                           -   -         -

Calculate enrichment
as likelihood ratio =              1   4         20
P(+) / P(-)
FunCoup prediction of 1 link
              Raw data
                Raw data
                 Raw data
                  Raw data
                    Raw data
          Bayesian LLR score
           Bayesian LLR score
            Bayesian LLR score
             Bayesian LLR score
              Bayesian LLR score

Sum of LLR scores

Confidence value
       pfc
Naïve Bayesian training
• Training:
   – Learn log likelihood ratios (LLRs) for each individual evidence bin
   – When predicting, sum all the LLRs to a full Bayesian score (FBS).


                    |ε |        P( Eij | FC )
      FBS (ε ) = ∑ log
                    i =1           P ( Eij )


      FC Functional coupling

      ε     Set of evidences
      Eij   Evidence i, bin j
4 training datasets → 4 different types
         of functional coupling
• Metabolic pathway
  (KEGG)



• Signalling pathway
  (KEGG)


• Physical protein-protein interaction


• Complex member
FunCoup training
                                                                                                                                               BAYESIAN FRAMEWORK

         INPUT DATA




107

                                                                       Human
105

                                                                 Rat
                                                                     Mouse
                                                                                           TRAINING SETS
 103                                                           Fly




                                                                         ×
       MEX                                                 Worm
             MIR
                   SCL                                   Yeast
                         PPI
                               PEX
                                     PHP               Plant
                                           TFB
                                                 DOM



                                                                                                                                                   ƒx, ƒy, ƒz, …
                                                                               25000
                                                                               20000
                                                                               15000
                                                                                10000                                                  Human
                                                                                 5000                                                 Mouse
                                                                                                                                 Rat
                                                                                                                                Fly
                                                                                        FC-PI
                                                                                                                           Worm
                                                                                                FC-CM
                                                                                                                         Yeast
                                                                                                        FC-ML
                                                                                                                FC-SL   Plant
Raw data metrics on CDC2 – KPNB1
  Fly   MEX (Li and White, 2003)            PLC=0.42
  Rat   MEX (Di Giovanni et al., 2004)      PLC=0.48
  Mouse SLC (UniProt, ESLDB)                WMI=0.04
  Mouse MEX (Zapala et al., 2005)           PLC=0.70
  Mouse MEX (Su et al., 2004)               PLC= -0.01
  Mouse MEX (Siddiqui et al., 2005)         PLC=0.56
  Mouse MEX (Hutton et al., 2004)           PLC=0.61
  Human PPI (IntAct, HPRD, BIND)            PPI score=0.17
  Human MEX (Su et al., 2004)               PLC=0.60
  …


                                                                  FC-SL model
                                                           FC-ML model
                                                      ΣSL =0+0-0.6+1.2-0.4+0.2+1.2+6.8+1.4=5.5
                                                    FC-CM model
                                               ΣSL =0+0-0.6+1.2-0.4+0.2+1.2+6.8+1.4=5.8
                                              FC-PI model
                                         ΣSL =0+0-0.6+1.2-0.4+0.2+1.2+6.8+1.4=7.9
                              FBSPI = 0+0-0.6+1.2-0.4+0.2+1.2+6.3+1.4…= 11.2




(pfc scores)
FBS score and pfc confidence
               |ε |           P ( Eij | FC )          FC Functional coupling
FBS (ε ) = ∑ log
               i =1              P ( Eij )            ε     Set of evidences
                                                      Eij   Evidence i, bin j




                                 |ε |
                      P( FC )∏ P( Eij | FC )
pfc(ε ) =              |ε |
                                 i =1
                                               |ε |
            P( FC )∏ P( Eij | FC ) + ∏ P( Eij )
                       i =1                    i =1
The total human FunCoup 2.0 network


Nr of links

5,000,000
4,500,000
4,000,000
3,500,000
3,000,000
2,500,000
2,000,000
1,500,000
1,000,000
  500,000
        0
              0.1   0.25    0.75
              Confidence cutoff
Nr of links at pfc cutoffs
          10000000



                                                                                     H. sapiens
                                                                                     M. musculus
           8000000
                                                                                     R. norvegicus
                                                                                     C. familiaris
                                                                                     D. rerio
           6000000
                                                                                     C. intestinalis
# links




                                                                                     D. melanogaster
                                                                                     C. elegans
                                                                                     G. gallus
           4000000                                                                   A. thaliana




           2000000




                 0
                     0   0.05   0.1   0.15 0.2   0.25 0.3   0.35   0.4   0.45 0.5   0.55 0.6   0.65   0.7   0.75 0.8   0.85 0.9   0.95
                                                                         pfc cutoff 
Comparison to STRING
• FunCoup on average 75% larger (based on all links)
 5000000



 4000000
                         FunCoup 2.0
                         STRING 9.0
 3000000



 2000000



 1000000



      0
                    C. elegans      C. intestinalis  D. rerio         H. sapiens     R. norvegicus
           A. thaliana      C. familiaris D. melanogaster     G. gallus       M. musculus     S. cerevisiae
Support from species and evidence type

                           MEX: mRNA co-expression

                           PHP: phylogenetic profile
                           similarity

                           PPI: protein–protein interaction

                           SCL: sub-cellular co-localization

                           MIR: co-miRNA regulation by
                           shared miRNA targeting

                           DOM: domain interactions

                           PEX: protein co-expression

                           TFB: shared transcription factor
                           binding

                           GIN: genetic interaction profile
                           similarity
Validation:
       Recovering cancer pathways
• 36 signalling links in
  RTK/RAS/PI(3)K, p53, and
  RB signalling pathways
  (TCGARN, Science 2008).

• FunCoup predicted 29 of 36
  links.

• 25 more links found.
Independent validation:
   Recovering tumour mutation sets
• Lists of genes co-mutated in glioblastoma tumours
  (The Cancer Genome Atlas).

• 6 of 9 lists (>= 10 genes) enriched (p<10-3) with
  internal FunCoup connections compared to random
  networks (preserving degree distribution).
FunCoup applications
           Find novel
           interactions




Extend                                     Find network
pathways                  FunCoup          modules




  Find novel
  disease genes                     Cross-talk
                                    between groups
http://FunCoup.sbc.su.se



                                                  ASPM




ASPM - Abnormal spindle-like microcephaly-associated protein
Data details
Klammer M, Roopra S, Sonnhammer EL. ”jSquid: a Java applet for
graphical on-line network exploration” Bioinformatics 2008, 24:1467
Comparative interactomics




New in FunCoup 2.0 – ensures true conservation
Human presenilin in worm
RNA-polymerase II subunits: yeast-all
Comparative interactomics
              Applications
• Hypothesis testing
   – Is a given pathway/complex conserved in another species?


• New discoveries
   – Finding ortholog pairs with conserved functional coupling – very
     strong evidence for functional conservation
   – Can also find conservation that is not strictly 4-way:

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NetBioSIG2012 eriksonnhammer

  • 1. Comparative Interactomics with FunCoup 2.0 Erik Sonnhammer Stockholm Bioinformatics Centre Science for Life Laboratory Dept. Biochemistry and Biophysics Stockholm University
  • 2. How to map the human interactome? • Genes: ~22000 • Interactions: 100000-300000? • Known direct interactions: ~74000 (Intact) • Experiments have high false negative and false positive rates. • → Most interactions need to be inferred combinatorially
  • 3. FunCoup: Predicting Functional Coupling Between Genes/Proteins Using Genomics Data and Orthology • Alexeyenko et al., NAR 40:D821 (2012) • Alexeyenko & Sonnhammer, Genome Research 19:1107 (2009)
  • 4. Co-expression patterns Phylogenetic profiles Domain interactions Shared transcription FunCoup Protein-protein interactions factor binding Shared Orthology Genetic Subcellular miRNA interactions co-localisation targeting Other Organisms
  • 5. Naïve Bayesian training -1.0 1.0 Continuous variable 0.6 1.0 Discrete categories Extract links + + + Test against positive and ”negative” reference datasets - - - Calculate enrichment as likelihood ratio = 1 4 20 P(+) / P(-)
  • 6. FunCoup prediction of 1 link Raw data Raw data Raw data Raw data Raw data Bayesian LLR score Bayesian LLR score Bayesian LLR score Bayesian LLR score Bayesian LLR score Sum of LLR scores Confidence value pfc
  • 7. Naïve Bayesian training • Training: – Learn log likelihood ratios (LLRs) for each individual evidence bin – When predicting, sum all the LLRs to a full Bayesian score (FBS). |ε | P( Eij | FC ) FBS (ε ) = ∑ log i =1 P ( Eij ) FC Functional coupling ε Set of evidences Eij Evidence i, bin j
  • 8. 4 training datasets → 4 different types of functional coupling • Metabolic pathway (KEGG) • Signalling pathway (KEGG) • Physical protein-protein interaction • Complex member
  • 9. FunCoup training BAYESIAN FRAMEWORK INPUT DATA 107 Human 105 Rat Mouse TRAINING SETS 103 Fly × MEX Worm MIR SCL Yeast PPI PEX PHP Plant TFB DOM ƒx, ƒy, ƒz, … 25000 20000 15000 10000 Human 5000 Mouse Rat Fly FC-PI Worm FC-CM Yeast FC-ML FC-SL Plant
  • 10. Raw data metrics on CDC2 – KPNB1 Fly MEX (Li and White, 2003) PLC=0.42 Rat MEX (Di Giovanni et al., 2004) PLC=0.48 Mouse SLC (UniProt, ESLDB) WMI=0.04 Mouse MEX (Zapala et al., 2005) PLC=0.70 Mouse MEX (Su et al., 2004) PLC= -0.01 Mouse MEX (Siddiqui et al., 2005) PLC=0.56 Mouse MEX (Hutton et al., 2004) PLC=0.61 Human PPI (IntAct, HPRD, BIND) PPI score=0.17 Human MEX (Su et al., 2004) PLC=0.60 … FC-SL model FC-ML model ΣSL =0+0-0.6+1.2-0.4+0.2+1.2+6.8+1.4=5.5 FC-CM model ΣSL =0+0-0.6+1.2-0.4+0.2+1.2+6.8+1.4=5.8 FC-PI model ΣSL =0+0-0.6+1.2-0.4+0.2+1.2+6.8+1.4=7.9 FBSPI = 0+0-0.6+1.2-0.4+0.2+1.2+6.3+1.4…= 11.2 (pfc scores)
  • 11. FBS score and pfc confidence |ε | P ( Eij | FC ) FC Functional coupling FBS (ε ) = ∑ log i =1 P ( Eij ) ε Set of evidences Eij Evidence i, bin j |ε | P( FC )∏ P( Eij | FC ) pfc(ε ) = |ε | i =1 |ε | P( FC )∏ P( Eij | FC ) + ∏ P( Eij ) i =1 i =1
  • 12. The total human FunCoup 2.0 network Nr of links 5,000,000 4,500,000 4,000,000 3,500,000 3,000,000 2,500,000 2,000,000 1,500,000 1,000,000 500,000 0 0.1 0.25 0.75 Confidence cutoff
  • 13. Nr of links at pfc cutoffs 10000000 H. sapiens M. musculus 8000000 R. norvegicus C. familiaris D. rerio 6000000 C. intestinalis # links D. melanogaster C. elegans G. gallus 4000000 A. thaliana 2000000 0 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 pfc cutoff 
  • 14. Comparison to STRING • FunCoup on average 75% larger (based on all links) 5000000 4000000 FunCoup 2.0 STRING 9.0 3000000 2000000 1000000 0 C. elegans C. intestinalis D. rerio H. sapiens R. norvegicus A. thaliana C. familiaris D. melanogaster G. gallus M. musculus S. cerevisiae
  • 15. Support from species and evidence type MEX: mRNA co-expression PHP: phylogenetic profile similarity PPI: protein–protein interaction SCL: sub-cellular co-localization MIR: co-miRNA regulation by shared miRNA targeting DOM: domain interactions PEX: protein co-expression TFB: shared transcription factor binding GIN: genetic interaction profile similarity
  • 16. Validation: Recovering cancer pathways • 36 signalling links in RTK/RAS/PI(3)K, p53, and RB signalling pathways (TCGARN, Science 2008). • FunCoup predicted 29 of 36 links. • 25 more links found.
  • 17. Independent validation: Recovering tumour mutation sets • Lists of genes co-mutated in glioblastoma tumours (The Cancer Genome Atlas). • 6 of 9 lists (>= 10 genes) enriched (p<10-3) with internal FunCoup connections compared to random networks (preserving degree distribution).
  • 18. FunCoup applications Find novel interactions Extend Find network pathways FunCoup modules Find novel disease genes Cross-talk between groups
  • 19. http://FunCoup.sbc.su.se ASPM ASPM - Abnormal spindle-like microcephaly-associated protein
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  • 22. Klammer M, Roopra S, Sonnhammer EL. ”jSquid: a Java applet for graphical on-line network exploration” Bioinformatics 2008, 24:1467
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  • 26. Comparative interactomics New in FunCoup 2.0 – ensures true conservation
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  • 31. Comparative interactomics Applications • Hypothesis testing – Is a given pathway/complex conserved in another species? • New discoveries – Finding ortholog pairs with conserved functional coupling – very strong evidence for functional conservation – Can also find conservation that is not strictly 4-way: