1. Bioinformatic Tools for Comparative Genomics of Vectors Comparative Genomics

2. Bioinformatic Tools for Comparative Genomics of Vectors Overview  Comparing Genomes  Homologies and Families  Sequence Alignments

3. Bioinformatic Tools for Comparative Genomics of Vectors Comparative Genomics  Allows us to achieve a greater understanding of vertebrate evolution  Tells us what is common and what is unique between different species at the genome level  The function of human genes and other regions may be revealed by studying their counterparts in lower organisms  Helps identify both coding and non-coding genes and regulatory elements

4. Bioinformatic Tools for Comparative Genomics of Vectors Sequence Conservation Over Time

5. Bioinformatic Tools for Comparative Genomics of Vectors  Large stretches of non-coding regions in vertebrates  Regulatory regions of: Developmental genes Transcription factors miRNA Non Coding Regions Kikuta et al., Genome Research, May 2007

6. Bioinformatic Tools for Comparative Genomics of Vectors Methods of Alignment- Ensembl  BLASTZ-net (comparison on nucleotide level) is used for species that are evolutionary close, e.g. human – mouse  Translated BLAT (comparison on amino acid level) is used for evolutionary more distant species, e.g. human – zebrafish  PECAN global alignment used for multispecies alignments

7. Bioinformatic Tools for Comparative Genomics of Vectors We can better understand evolution/ speciation We can find important, functional regions of the sequence (codons, promoters, regulatory regions) It can help us locate genes in other species that are missing or not well- defined (also through comparison and alignments). Quality control! Why Compare Genomes?

8. Bioinformatic Tools for Comparative Genomics of Vectors Evolution at the DNA Level …ACTGACATGTACCA… …AC----CATGCACCA… Mutation Sequence edits Rearrangements Deletion Inversion Translocation Duplication

9. Bioinformatic Tools for Comparative Genomics of Vectors Mammals have roughly 3 billion base pairs in their genomes Over 98% human genes are shared with primates, with more than 95- 98% similarity between genes. Even the fruit fly shares 60% of its genes with humans! (March 2000) Compare human & Mouse 40% of human genome align with mouse 24% of human genome missing in mouse (also mouse-specific sequences) Comparing Genomes

10. Bioinformatic Tools for Comparative Genomics of Vectors Improving Gene Quality Comparative genomics predicts one long transcript.

11. Bioinformatic Tools for Comparative Genomics of Vectors Pseudogene recovery chr 3chr X human mouse rat dog cow We find 67 confident cases where a human protein is closer to the ancestor than any extant species in the alignment

12. Bioinformatic Tools for Comparative Genomics of Vectors Uses all the species Prediction pipeline: Begins with BLAST and sequence clustering Compares gene relationships to species relationships How Does Ensembl Predict Homology?

13. BSR: Blast Score Ratio. When 2 proteins P1 and P2 are compared, BSR=scoreP1P2/max(self- scoreP1 or self-scoreP2). The default threshold used in the initial clustering step is 0.33.

14. Bioinformatic Tools for Comparative Genomics of Vectors Orthologue / Paralogue Prediction Algorithm (1) Load the longest translation of each gene from all species used in Ensembl. (2) Run WUBLASTp+SW of every gene against every other (both self and non-self species) in a genome-wide manner. (3) Build a graph of gene relations based on Best Reciprocal Hits (BRH) and Blast Score Ratio (BSR) values. (4) Extract the connected components (=single linkage clusters), each cluster representing a gene family. (5) For each cluster, build a multiple alignment based on the protein sequences using MUSCLE. (6) For each aligned cluster, build a phylogenetic tree using PHYML. An unrooted tree is obtained at this stage. (7) Reconcile each gene tree with the species tree to call duplication event on internal nodes and root the tree (TreeBeSt). (8) From each gene tree, infer gene pairwise relations of orthology and paralogy types.

15. Bioinformatic Tools for Comparative Genomics of Vectors Species Tree

16. Bioinformatic Tools for Comparative Genomics of Vectors Phylogenetic Tree Reconciliation: the Species/Gene Tree Problem Dufayard et al. ERCIM News No. 43 October 2000 Species and Gene Trees

17. Bioinformatic Tools for Comparative Genomics of Vectors Genes/Species Tree reconciliation: TreeBeST

18. Reconciliation M R H M R H species tree unrooted gene tree Duplication node Speciation node MRHMRH MHRMHR gene loss R’ H’ M’

19. Bioinformatic Tools for Comparative Genomics of Vectors Viewing Trees in Ensembl  GeneView page  GeneTreeView

20. Bioinformatic Tools for Comparative Genomics of Vectors Types of Homologues Orthologs : any gene pairwise relation where the ancestor node is a speciation event Paralogs : any gene pairwise relation where the ancestor node is a duplication event

21. Bioinformatic Tools for Comparative Genomics of Vectors Orthologue and Paralogue Types  ortholog_one2one  ortholog_one2many  ortholog_many2many  apparent_ortholog_one2one  within_species_paralog  between_species_paralog

22. Ortholog and Paralog types

23. Bioinformatic Tools for Comparative Genomics of Vectors Ortholog and Paralog types

24. Bioinformatic Tools for Comparative Genomics of Vectors What is ‘1 to 1’? What is ‘1 to many’? Orthologues on GeneView

25. Bioinformatic Tools for Comparative Genomics of Vectors Protein Families  How: Cluster proteins for every isoform (transcript) in every species.  Why: Predict a function for ‘novel’ genes/proteins Understand gene relationships

26. Bioinformatic Tools for Comparative Genomics of Vectors Protein Dataset More than 1,800,000 proteins clustered:  All Ensembl protein predictions from all species supported 895,070 protein predictions  All metazoan (animal) proteins in UniProt: 96,030 UniProtKB/Swiss-Prot 892,0208 UniProtKB/TrEMBL

27. Bioinformatic Tools for Comparative Genomics of Vectors Clustering Strategy  BLASTP all-versus-all comparison  Markov clustering  For each cluster:  Calculation of multiple sequence alignments with ClustalW  Assignment of a consensus description

28. Bioinformatic Tools for Comparative Genomics of Vectors Link to FamilyView Where are Families shown? ProtView

29. Bioinformatic Tools for Comparative Genomics of Vectors Ensembl family members within human Ensembl family members in other species JalView multiple alignments Where are Families shown? FamilyView

30. Bioinformatic Tools for Comparative Genomics of Vectors  Comparing Genomes  Homologies and Families  Sequence alignments

31. Bioinformatic Tools for Comparative Genomics of Vectors To identify homologous regions To spot trouble gene predictions Conserved regions could be functional To define syntenic regions (long regions of DNA sequences where order and orientation is highly conserved) Aligning Whole Genomes- Why?

32. Bioinformatic Tools for Comparative Genomics of Vectors  Should find all highly similar regions between two sequences  Should allow for segments without similarity, rearrangements etc.  Issues  Heavy process  Scalability, as more and more genomes are sequenced  Time constraint Aligning large genomic sequences

33.  Enredo  Defines orthology map (co-linear regions)‏  Supports segmental duplications  Pecan  Consistency based multiple aligner  Optimized to cope with long DNA sequences  Ortheus  Ancestral sequences reconstructor  Inferring the history of insertion and deletions Whole Genome Multiple Alignments

34. Bioinformatic Tools for Comparative Genomics of Vectors In ContigView...

35. Bioinformatic Tools for Comparative Genomics of Vectors  Currently 2 sets:  10 amniota vertebrates:  7 eutherian mammals: Multiple Alignments using PECAN To come… the fish!

36. Bioinformatic Tools for Comparative Genomics of Vectors  Use all coding exons  Get sets of best reciprocal hits  Create orthology maps  Use all coding exons  Get sets of best reciprocal hits  Create orthology maps  Build multiple global alignments Alignment Strategy  Use all coding exons  Get sets of best reciprocal hits

37. Bioinformatic Tools for Comparative Genomics of Vectors View Alignments: ContigView In the Detailed View Panel:

38. Bioinformatic Tools for Comparative Genomics of Vectors View Conservation: ContigView Click on a Pink Bar for AlignSliceView… export alignments

39. Bioinformatic Tools for Comparative Genomics of Vectors AlignSliceView

40. GeneSeqalignView

41. GeneSeqalignView

42. MultiContigView Comparison of chromosomes in multiple species. (Links from SyntenyView, ContigView, CytoView)

43. Bioinformatic Tools for Comparative Genomics of Vectors Export Alignments in BioMart Choose ‘Compara pairwise alignments’

44. Bioinformatic Tools for Comparative Genomics of Vectors Syntenic Regions  Genome alignments are compiled into larger syntenic regions  Alignments are clustered together when the relative distance between them is less than 100 kb and order and orientation are consistent  Any clusters less than 100 kb are discarded

45. Bioinformatic Tools for Comparative Genomics of Vectors Enredo Anchors 500.000 anchors for mammals --- more than 1 anchor per 10Kb Supports segmental duplications!! Covers 90% of the human protein coding genes (Hsap-Mmus-Rnor-Cfam-Btau)‏

46. Bioinformatic Tools for Comparative Genomics of Vectors SyntenyView Human chromosome Mouse chromosomes Orthologues

47. Bioinformatic Tools for Comparative Genomics of Vectors Syntenic blocks CytoView

48. Bioinformatic Tools for Comparative Genomics of Vectors Summary  View Homology in pages such as GeneView, ProtView, SyntenyView, GeneTreeView, or BioMart  View Protein Family information in FamilyView  View Alignments in ContigView, GeneSeqAlign View, through BioMart