Bioinformatics Bioinf and E ormatics volu and E tionar volu y Genomics Genom 5/2/2011 1

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Jessie Lamb
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1 Bioinformatics and Evolutionary Genomics 5/2/2011 1

2 Fritz-Laylin et al. cell 2010

3 Gene Trees, Gene Duplications, and Orthology How to make trees Bootstrap Interpreting trees duplications vs speciations vs loss, timing of duplications, HGT Orthology Duplications before LECA Endosymbiosis

4 Phylogenetic gene trees: how to make them Homology: are two pieces of sequence related; Trees: when did they diverge (how are they related) Start from a multiple sequence alignment All multiple sequence programs alignments make a global alignment, thus feed it regions that you know are homologous Domains! MUSCLE / clustal / t_coffee /MAFFT Visual inspection of alignments (gaps, fragments/complete sequences, weird things e.g. A)

5 Put homologs in the alignment Even if they are not homologous alignment programs will align them (muscle/clustal/mafft implicitly assumes that the sequences you feed it are homologous) And in a phylogeny program, non-homologous sequences will be clustered

6 Visual inspection of alignments:?!

7 An additive tree which is wrongly reconstructed by UPGMA A A 6 6 C C B B 5 2 D D C D B A A B C D A x B 12 x 9 7 C 9 9 x 6 D x A B CD A x 12 9 B 12 x 8 CD 9 8 x A BCD A x 10 BCD 10 x

8 Neighbour-Joining (Saitou and Nei, 1987) Global measure. keeps total branch length minimal At each step, join two nodes such that distances are minimal (criterion of minimal evolution) Leads to unrooted tree

9 Neighbour-Joining At each step all possible neighbour joinings are checked and the one corresponding to the minimal total tree length (calculated by adding all branch lengths) is taken.

10 A B C D r A x B 12 x C 9 9 x 6 24 D x 22 d au = d ac /2 + (r a -r c )/(2(N-2)) = 9/2 + (30-24)/(2*2) = 6 Neighbour-Joining M ab = d ab (r a +r b )/(N-2) Mab = 12 (30+28)/(4-2)) = -17 AC U A B C D A x B x C x -17 D x r= net divergence d cu = d ac - d au = 9 6 = 3 d bu = (d ab + d bc d ac ) / 2 = ( ) / 2 = 6 d du = (d ad + d cd d ac ) / 2 = (9+ 6 9) / 2 = 3 A 6 6 B C U 3 3 D

11 U B D r U x B 6 x 7 13 D 3 7 x 10 U B D U x B x -16 D x e.g. UB V D vu = d ub / 2 + (r u r b )/ (2(N-2)) = 6/2 + (9-13)/(2*1) = 3 2 = 1 D vb = d ub d uv = 6 1 = 5 D dv = (d ud +d bd d ub )/2 = (3+7-6)/2 = 2 A 6 U 1 V B 5 2 D 3 C

12 Unequal rates between species are a very real phenomenon

13 Character based: parsimony and maximum likelihood Two way classification in phylogeny distance based vs character based character state method. Searches directly (i.e. without defining distances) for a tree that fits best to the data (the alignment)

14 Maximum likelihood have to specify a model of sequence evolution likelihood for all sites is the product of the likelihoods for individual sites assuming all the sites evolve independently. maximum likelihood method computes the probabilities for all possible combinations of ancestral states! ML methods evaluate phylogenetic hypotheses in terms of the probability that a proposed model of the evolutionary process and the proposed unrooted tree (hypothesis) would give rise to the observed data (the alignment). The tree found to have the highest (log)ml value is considered to be the preferred tree. Currently: RaxML and WAG

15 Gene Trees, Gene Duplications, and Orthology How to make trees Bootstrap Interpreting trees duplications vs speciations vs loss, timing of duplications, HGT Orthology Duplications before LECA Endosymbiosis

16 How to assess confidence in tree bootstrap: Select multiple alignment columns with replacement Recalculate tree Compare branches with original tree Repeat times, so calculate different trees How often is branching preserved for each internal node? Uses samples of the data

17 The Bootstrap

18 The Bootstrap Original C C V K V I Y S M A V R L I F S M C L R L L F T Resampled V K V S I I S I V R V S I I S I L R L T L L T L 2x 1x 3x 2x Nonsupportive

19 The Bootstrap times times

20 Gene Trees, Gene Duplications, and Orthology How to make trees Bootstrap Interpreting trees duplications vs speciations vs loss, timing of duplications, HGT Orthology Duplications before LECA Endosymbiosis

21 Phylogenetic tree: historical pattern of relationships among organisms: interpretation of a tree Divergence (= speciation) between skunk and otter Ancestor of all extant carnivores NB still no information in skunk left / otter right

22 Interpreting the tree Taxonomic findings Paraphyly Monophyly

23 Simple example (kinase) What are the nodes?

24 Gene Trees, Gene Duplications, and Orthology How to make trees Bootstrap Interpreting trees duplications vs speciations vs loss, timing of duplications, HGT Orthology Duplications before LECA Endosymbiosis

25 Two genes per species: how to differentiate between one ancient or two recent duplications? Two genes in Human chromosomes (human A & Human B) & two genes in mouse chromosomes (Mouse A & Mouse B)

26 Duplications, Speciations 1 2 3?

27 Interpreting the tree: duplications vs speciations, going pseudo 3D Speciation

28 Interpreting the tree: gene trees vs species trees

29 Introduce a root root time Drosophila edge fugu mouse human root edge leaf internal node internal node (ancestor) leaf

30 How to root a tree: outgroup

31 Interpreting the tree Outgroup. place root between distant homologouss sequence and rest group (b) Midpoint. place root at midpoint of longest path (sum of branches between any two leafs) NB njplot Gene duplication. Place root between paralogous gene copies (b) NB all affected by rates! b

32 three kingdoms LUCA

33 How to root the tree of life? 1: Find paralogs that duplicated before the LUCA 6 found so far

34 How to root the tree of life? 2: Make a tree of paralogs that duplicated before the LUCA Griblado 1998 J Mol Evol

35 Interpreting the tree Example: vertebrate duplications Tetraploidy?

36 36 B

37 Interpreting the tree: Horizontal Gene Transfer ( HGT ) Bacteria Archaea Eukarya

38 Gene Trees, Gene Duplications, and Orthology How to make trees Bootstrap Interpreting trees duplications vs speciations vs loss, timing of duplications, HGT Orthology Duplications before LECA Endosymbiosis

39 Jargon for interpretation: Orthology (and paralogy) as a specification of homology when discussing two species human1 mouse1 human2 Fitch 1970 Two genes in two species are orthologous if they derive from a single gene in their last common ancestor the corresponding gene Genes can diverge by Speciation, or Duplication last common node is a speciation node implied to have the same function

40 Orthology ~ annotating internal nodes as duplications or speciations Because of the definition, how does that translate to a tree With or without species phylogeny? B

41 Terminology: inparalogs, outparalogs, co-orthologsorthologs Inparalogs Co-orthologs Outparalogs

42 Importance of orthology for comparative genomics: more resolution Ec Hi Bs Af Af Ec Bs Mg Gene family present in Ec Hi Bs Mg Af Orthologs 1 present in Ec Hi Bs Af Orthologs 2 present in Ec Bs Mg Af Phenotype ~ gene correlation Func prediction if Hi is only biochem characterized enzyme Func prediction by co-ococ Evolution of gene content: loss vs dupl

43 function prediction Blast with a newly sequenced globin from frog What kind of globin is it?

44 Blast query Globins

45 Blast query Globins

46 Orthologous & function prediction vs homologous that are not orthologous & function Orthologs tend to have the exact same molecular function, mere HTANO s not and operate in the same pathway. Orthologs mostly have the same domain composition; (exceptions e.g. fusion, fission)

47 but inparalogs: fate after duplication: neofunctionalization or subfunctionalization Even evolutionary true orthologs can have different functions Both co-orthologs have taken over some aspect of the ancestral function and have lost other aspects Acquiring of new function or loss-of-function: one of co-orthologs does something different now.

48 subfunctionalization: example in terms of protein complexes (=GO cellular component)

49 neofunctionalization: example in terms of protein complexes (=GO cellular component)

50 Sub vs neo in regulatory context OLD VIEW NEW VIEW Moore and Purugganan 2005 b

51 Methods to go from trees to orthologs First: methods that were strict (like I explained in the previous section) (blackboard) Currently: programs that take uncertainty into account and also weigh the amount of genome evolution that a topology implies NOTUNG, TREEBEST, SYNERGY

52 NOTUNG Use bootstrap ensemble (and minimum bootstrap value above which a clustering cannot be overridden): E.g. blackboard

53 Orthologs can have different domain composition Van Dam et al. 2009

54 Does retaining the ancestral role correlate with speed of sequence evolution: yes but a substantial minority is inconsistent

55 rfbb / rffg RfbB and RffG catalyze the same reaction, but are involved in two different biological processes. rfb gene cluster: biosynthesis of O-specific polysaccharides (inner membrane). rff gene cluster: complex biosynthesis of enterobacteria common antigen (outer membrane).

56 Frequency (# cases) Why inconsistencies? Consistent Inconsistent Sequence identity between inparalogs (%) Not because of chance due to lack of divergence time

57 Why do observe inconsistencies? Similar sequence divergence of inparalogs relative to their single- ortholog, molecular function similar? Any inconsistencies are then a chance outcome: both duplicates have diverged, but at (roughly) the same evolutionary speed (most amino acids substitutions are only been subject to purifying selection and not to adaptive selection)

58 In certain orthology scheme gene order is given prevalence above most similarity Gene at conserved position is considered the original and the other duplicate the copy

59 Genome alignments help (a.o.).) to more detailed reconstruction Human h_a h_a h_a h_a m_a Mouse m_a

60 Gene Trees, Gene Duplications, and Orthology How to make trees Bootstrap Interpreting trees duplications vs speciations vs loss, timing of duplications, HGT Orthology Duplications before LECA Endosymbiosis

61 Gene Trees allow to look at events from before LECA Lack of descendants of intermediates

62 Feca/eocyteeocyte to LECA Nucleic Acids Res Aug 16;33(14): Ancestral paralogs and pseudoparalogs and their role in the emergence of the eukaryotic cell. Makarova KS, Wolf YI, Mekhedov SL, Mirkin BG, Koonin EV.

63 duplications: e.g. small GTPases

64 RHIORY04768 Rhizopus oryzae RO3G RHIORY12029 Rhizopus oryzae RO3G RHIORY08782 Rhizopus oryzae RO3G PHYBLA07488 Phycomyces blakesleeanus PHYBLA10547 Phycomyces blakesleeanus CIOINT11638 Ciona intestinalis ENSCINP NEMVEC07636 Nematostella vectensis DANPUL03030 Daphnia pulex ANOGAM07785 Anopheles gambiae AGAP PA 45 STRPUR01391 Strongylocentrotus purpuratus FUGRUB07510 Fugu rubripes ENSTRUP GALGAL16219 Gallus gallus ENSGALP HOMSAP46439 Homo sapiens ENSP SIPA1L2 MUSMUS18957 Mus musculus ENSMUSP ORNANA04487 Ornithorhynchus anatinus ENSOANP DANRER14764 Danio rerio ENSDARP FUGRUB42991 Fugu rubripes ENSTRUP ORNANA07813 Ornithorhynchus anatinus ENSOANP HOMSAP05420 Homo sapiens ENSP SIPA1L3 MUSMUS21980 Mus musculus ENSMUSP FUGRUB25077 Fugu rubripes ENSTRUP DANRER25213 Danio rerio ENSDARP FUGRUB38972 Fugu rubripes ENSTRUP GALGAL11004 Gallus gallus ENSGALP ORNANA13730 Ornithorhynchus anatinus ENSOANP XENTRO15457 Xenopus tropicalis ENSXETP HOMSAP13282 Homo sapiens ENSP SIPA1L1 MUSMUS08282 Mus musculus ENSMUSP FUGRUB44440 Fugu rubripes ENSTRUP MUSMUS00454 Mus musculus ENSMUSP HOMSAP18141 Homo sapiens ENSP SIPA1 CAEELE24679 Caenorhabditis elegans T27F2.2 TRIADH06340 Trichoplax adhaerens MONBRE08116 Monosiga brevicollis BRAFLO11027 Branchiostoma floridae BRAFLO39172 Branchiostoma floridae DANPUL04258 Daphnia pulex ANOGAM10606 Anopheles gambiae AGAP PA DROMEL06806 Drosophila melanogaster FBpp XENTRO18986 Xenopus tropicalis ENSXETP GALGAL02952 Gallus gallus ENSGALP MUSMUS10583 Mus musculus ENSMUSP HOMSAP07367 Homo sapiens ENSP GARNL4 ORNANA16509 Ornithorhynchus anatinus ENSOANP FUGRUB13197 Fugu rubripes ENSTRUP DANRER12772 Danio rerio ENSDARP ORNANA12366 Ornithorhynchus anatinus ENSOANP DANRER11514 Danio rerio ENSDARP ORNANA11599 Ornithorhynchus anatinus ENSOANP HOMSAP41753 Homo sapiens ENSP RAP1GAP DANRER26558 Danio rerio ENSDARP FUGRUB30853 Fugu rubripes ENSTRUP FUGRUB33582 Fugu rubripes ENSTRUP GALGAL20607 Gallus gallus ENSGALP FUGRUB04867 Fugu rubripes ENSTRUP CIOINT17316 Ciona intestinalis ENSCINP LOTGIG02597 Lottia gigantea CAEELE08395 Caenorhabditis elegans F53A10.2c BRAFLO03033 Branchiostoma floridae DANPUL13737 Daphnia pulex DANPUL13734 Daphnia pulex ANOGAM08799 Anopheles gambiae AGAP PA DROMEL09545 Drosophila melanogaster FBpp NEMVEC09465 Nematostella vectensis TRIADH03922 Trichoplax adhaerens BATDEN03327 Batrachochytrium dendrobatidis BDEG DICDIS10986 Dictyostelium discoideum DDB DICDIS09075 Dictyostelium discoideum DDB DICDIS10992 Dictyostelium discoideum DDB DICDIS10990 Dictyostelium discoideum DDB DICDIS10993 Dictyostelium discoideum DDB DICDIS10987 Dictyostelium discoideum DDB DICDIS10988 Dictyostelium discoideum DDB DICDIS10462 Dictyostelium discoideum DDB FUGRUB33128 Fugu rubripes ENSTRUP DANRER30794 Danio rerio ENSDARP XENTRO19451 Xenopus tropicalis ENSXETP GALGAL03829 Gallus gallus ENSGALP ORNANA19644 Ornithorhynchus anatinus ENSOANP HOMSAP23130 Homo sapiens ENSP GARNL3 MUSMUS34198 Mus musculus ENSMUSP BRAFLO46730 Branchiostoma floridae BRAFLO38475 Branchiostoma floridae LOTGIG03108 Lottia gigantea DANPUL05445 Daphnia pulex ANOGAM11069 Anopheles gambiae AGAP PA TRIADH07590 Trichoplax adhaerens MONBRE00371 Monosiga brevicollis 5147 BATDEN00833 Batrachochytrium dendrobatidis BDEG PHYSOJ14061 Phytophthora sojae PHYINF15173 Phytophthora infestans PITG NAEGRU04462 Naegleria gruberi PHYSOJ09210 Phytophthora sojae PHYINF03903 Phytophthora infestans PITG BRAFLO25710 Branchiostoma floridae BRAFLO48761 Branchiostoma floridae DANRER26667 Danio rerio ENSDARP FUGRUB22220 Fugu rubripes ENSTRUP GALGAL04384 Gallus gallus ENSGALP MUSMUS36814 Mus musculus ENSMUSP HOMSAP03383 Homo sapiens ENSP KIAA ORNANA20556 Ornithorhynchus anatinus ENSOANP XENTRO24458 Xenopus tropicalis ENSXETP LOTGIG22079 Lottia gigantea STRPUR10477 Strongylocentrotus purpuratus DANPUL21435 Daphnia pulex DROMEL06371 Drosophila melanogaster FBpp NEMVEC06293 Nematostella vectensis CAEELE10980 Caenorhabditis elegans D2085.5a BATDEN08289 Batrachochytrium dendrobatidis BDEG RHIORY08780 Rhizopus oryzae RO3G PHYBLA13159 Phycomyces blakesleeanus DICDIS10480 Dictyostelium discoideum DDB CIOINT14237 Ciona intestinalis ENSCINP BRAFLO28054 Branchiostoma floridae BRAFLO40792 Branchiostoma floridae ORNANA15244 Ornithorhynchus anatinus ENSOANP HOMSAP02932 Homo sapiens ENSP C20orf MUSMUS36384 Mus musculus ENSMUSP XENTRO10193 Xenopus tropicalis ENSXETP GALGAL15615 Gallus gallus ENSGALP FUGRUB38803 Fugu rubripes ENSTRUP DANRER17001 Danio rerio ENSDARP ORNANA26082 Ornithorhynchus anatinus ENSOANP GALGAL11140 Gallus gallus ENSGALP MUSMUS08042 Mus musculus ENSMUSP HOMSAP12892 Homo sapiens ENSP GARNL1 48 XENTRO18775 Xenopus tropicalis ENSXETP FUGRUB05413 Fugu rubripes ENSTRUP NEMVEC15188 Nematostella vectensis DANPUL15969 Daphnia pulex ANOGAM12607 Anopheles gambiae AGAP PA DROMEL19986 Drosophila melanogaster FBpp LOTGIG18914 Lottia gigantea STRPUR17268 Strongylocentrotus purpuratus STRPUR17221 Strongylocentrotus purpuratus MONBRE08208 Monosiga brevicollis CAEELE04065 Caenorhabditis elegans Y18H1A.3 51 RHIORY15087 Rhizopus oryzae RO3G PHYBLA07175 Phycomyces blakesleeanus BATDEN04658 Batrachochytrium dendrobatidis BDEG DICDIS10991 Dictyostelium discoideum DDB NAEGRU15224 Naegleria gruberi CYAMER01669 Cyanidioschyzon merolae CMK017C BATDEN00532 Batrachochytrium dendrobatidis BDEG USTMAY00939 Ustilago maydis UM CRYNEO02908 Cryptococcus neoformans CNAG PHACHR06765 Phanerochaete chrysosporium RHIORY03220 Rhizopus oryzae RO3G YARLIP03962 Yarrowia lipolytica YALI0E07095g KLULAC00510 Kluyveromyces lactis KLLA0A11792g 64 CANGUI05373 Candida guilliermondii PGUG DEBHAN01781 Debaryomyces hansenii DEHA0C07986g 83 SCHPOM02683 Schizosaccharomyces pombe SPAC630.13c NEUCRA03708 Neurospora crassa NCU04105 FUSGRA06737 Fusarium graminearum FGSG SCLSCL00568 Sclerotinia sclerotiorum SS1G COCIMM08406 Coccidioides immitis CIMG ASPTER07029 Aspergillus terreus ATEG PHATRI05157 Phaeodactylum tricornutum MONBRE09131 Monosiga brevicollis DANPUL00443 Daphnia pulex ANOGAM01899 Anopheles gambiae AGAP PA DROMEL15313 Drosophila melanogaster FBpp LOTGIG00442 Lottia gigantea BRAFLO01835 Branchiostoma floridae FUGRUB15678 Fugu rubripes ENSTRUP XENTRO25350 Xenopus tropicalis ENSXETP ORNANA04688 Ornithorhynchus anatinus ENSOANP GALGAL05064 Gallus gallus ENSGALP HOMSAP09816 Homo sapiens ENSP TSC2 MUSMUS02602 Mus musculus ENSMUSP CIOINT06080 Ciona intestinalis ENSCINP STRPUR02821 Strongylocentrotus purpuratus DICDIS10989 Dictyostelium discoideum DDB PHYINF03733 Phytophthora infestans PITG TETTHE05210 Tetrahymena thermophila 6730 Rap/Ral/RheBRheB GAP tree: events from before the LECA i.e. eukaryotes from all over the tree of life scattered throughout the tree 0.5

65 Rap/Ral/RheBRheB GAP tree: events from before the LECA 100 RapGAP (animals(lse), fungi, dicty) PHYSOJ14061 Phytophthora sojae PHYINF15173 Phytophthora infestans PITG RalGAPB (oomycetes, dicty, naegleria, fungi, animals)) 23 8 RalGAPA (dicty, naegleria, fungi, animals) 24 RheBGAP (TSC2, oomycetes, diatoms, red algea, animals, fungi, dicty, tetrahymena) 0.5

66 Gene Trees, Gene Duplications, and Orthology How to make trees Bootstrap Interpreting trees duplications vs speciations vs loss, timing of duplications, HGT Orthology Duplications before LECA Endosymbiosis

67 Endo symbiosis of alpha proteo-bacteria gave rise to mitochondria Mitochondrial DNA in the mitochondria Hydrogenosomes shown to be derived from mitochondria Many proteins active in present-day mitochondria are coded for by proteins of eukaryotic invention, archaeal descent Many proteins of alpha-protein ancestor active in in other parts of the cell B

68 rrna tree Mitochondria have their own mini genome 16S Ribosomal RNA

69 Identifying eukaryotic proteins with an alpha- proteobacterial origin based on their phylogeny Eukaryotic + alpha-proteobacteria in the same branch Alpha-proteobacterial proteins with the rest of the bacteria and archaea

70 Detecting eukaryotic genes of alpha-proteobacterialproteobacterial ancestry GENOME 6 alpha-proteobacteria ( genes) GENOMES 6 alpha-proteobacteria 9 eukaryotes 56 Bacteria+Archaea SELECTION OF HOMOLOGS, (Smith&Waterman) LIST ALIGNMENTS AND TREE (Clustalx, Kimura+Dayhoff) TREE SCANNING PHYLOME

71 Benchmarking? 1. a control 2. ML works!

72 Proto-mitochondrial metabolism: - Catabolism of fatty acids, glycerol and amino acids. - Some pathways are not mitochondrial. non-mitoch.. mitochondrial not in yeast/human

73 The majority of the proto-mitochondrial proteome is not mitochondrial (anymore) Yeast mitochondrial proteome: Eric Schon, Methods Cell Biol 2001 (manually curated) Gabaldon & Huynen Science 2003 alpha-prot Huh et al., Nature 2003 (green fluorescent genomics) 527 Human mitochondrial proteome: 755 Eric Schon, Methods Cell Biol

74 From endosymbiont to organell, not only loss and gain of proteins but also retargeting : proteins Ancestor loss gain re-targeting t ~65% of the alphaproteobacteria derived set is not mitochondrial. Modern mitochondria ~16% of the mitochondrial yeast proteins are of alphaproteobacterial origin. Gabaldon and Huynen, Science 2004

Elements of Bioinformatics 14F01 TP5 -Phylogenetic analysis

Elements of Bioinformatics 14F01 TP5 -Phylogenetic analysis 10 December 2012 - Corrections - Exercise 1 Non-vertebrate chordates generally possess 2 homologs, vertebrates 3 or more gene copies; a Drosophila