Orthology & Paralogy Alignment & Assembly Alastair Kerr Ph.D. WTCCB Bioinformatics Core [many slides borrowed from various sources]

Overview  Orthology & Paralogy Definitions and examples Ways to determine an ortholog Pre-calculations: resources  Alignment & Assembly Differences Key programs for each Jalview example

Homologs Have common origins but may or may not have common activity. Homologous or not?: Often determined by arbitrary threshold level of similarity determined by alignment

Homologs …have common ancestry, but the way they are related can vary (i.e. the reasons they have diverged into different sequences can vary)  orthologs - Homologs produced by speciation. They tend to have similar function.  paralogs - Homologs produced by gene duplication. They tend to have differing functions.

Orthologous or paralogous homologs Early globin gene mouse  ß -chain gene  -chain gene cattle ß human ß mouse ß human  cattle  Orthologs (  ) Orthologs ( ß ) Paralogs (cattle) Homologs Gene Duplication Orthologs – diverged after speciation – tend to have similar function Paralogs – diverged after gene duplication – some functional divergence occurs Therefore, for linking similar genes between species, or performing “annotation transfer”, identify orthologs

True or False? A1x is the ortholog in species x of A1y? A1x is a paralog of A2x? A1x is a paralog of A2y?

Identifying Gene/Protein Relationships from Phylogenies  Orthologs – Homologs produced by speciation – Gene phylogeny matches organismal phylogeny  Paralogs – Homologs produced by gene duplication. – Multiple copies of homologs in a given species or evidence that gene duplication involved through phylogenetic analysis – Lack of match to organismal phylogeny

Gene Orthology: How to detect?  Most : Identify reciprocal best BLAST hits (EGO, COGs,…) Example Problem:  If making comparisons between human and bovine, for example, the bovine gene dataset is still quite incomplete  Therefore, current best hit may be a paralog now and the true ortholog not yet sequenced cattlehumancattle mouse

2 Forms in 1 Species ++++ Slides from Jonathan Eisen

2 Forms in 1 Species - Gene Loss Gene duplicated in common ancestor Loss

Unusual Distribution Pattern + +

Unusual Distribution - Gene Loss + + Gene present in ancestor Gene lost here

Unusual Distribution - Evolutionary Rate Variation -? + + Gene too diverged to be found

Ortholog guess via synteny ACB AC?

Syntenic blocks

Alignments and Assemblies  Alignment ALL sequences from SAME region Therefore can be useless for  non-overlapping contigs  PCR probes/oligos Good for  paralog/orthologs  Basis for phylogeny  More dissimilar sequences  Assembly: Good for near identical sequences Read Length  Short Read [Next Gen Sequencing]  Long Read [Sanger and 3 rd Gen sequencing?] Reference?  De-novo  Guided [reference sequence]

ensEMBL calculations demo

OMA Browser demo

Alignment  Implicit statement Each residue in an aligned sequence derived from the last common ancestor [LCA]  Therefore ok to only look at conserved regions or mask non- conserved regions Especially for phylogeny

Alignment Tools  Faster but less accurate (some better with gaps) Muscle ClustalW/X MAFFT  Slow but more accurate *-Coffee  T: original  3D: uses pdb as guide (structural)  M: uses multiple methods Probcons

Alignment Edit Tools  NEVER use a word processor or excel to edit alignments……  JalView (Java Alignment Viewer) Good for editing DAS capable

Figure Generation Trees Annotation Features Structures PDB ‘Standard’ Formats FASTA MSF CLUSTAL PILEUP BLC PFAM Distributed Annotation System Distributed Annotation System GFF Jalview Features Newick Secondary Structure Prediction Multiple Sequence Alignment Sequences Alignments Clickable HTML Images Line Art Analysis Consensus Conservation & Clustering Visualization Jalview Annotation

Jalview DAS Client Functionality DAS ANNOTATION SERVERS DAS ANNOTATION SERVERS Query matches ID to Authority Map to local reference frame Mouse over for feature name, links and scores Group features by source Type==colour Highlight start-end Select specific sources Filtered list Add user defined sources

Assemblers  Many free options : examples below  Long Reads STADEN - staden.sf.net  NextGenSequencing Guided: Bowtie, Novoalign, MAQ Denovo: Velvet  3 rd Generation Sequencing ????

Post Assembly  Correction Reads mapping to multiple places PCR amplification prior to mapping  Tools and workflows available in our Galaxy platform demo