Comparative Genomics. Bing Zhang. Department of Biomedical Informatics Vanderbilt University

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1 Comparative Genomics Bing Zhang Department of Biomedical Informatics Vanderbilt University

2 Genome and genome project Genome: the complete genetic material of an organism. E. coli genome: 4.7 million base pairs of DNA Human genome: 3 billion base pairs of DNA (~3G) Encodes heritable characteristics Genome project: scientific endeavors that ultimately aim to determine the complete genome sequence of an organism 2

3 Completely sequenced genomes Human genome project 3

4 A gene for truckers Genome function Microarray RNA-seq Proteomics Comparative genomics 4

5 Comparative genomics Comparative genomics Analysis and comparison of genomes from different species Underlying hypothesis Genomes under examination had a common ancestor and, therefore, every base pair in each organism can be explained as the combination of this original ancestral genome and the action of evolution Applications Relationship among species Functional sequences 5

6 Genome evolution and functional sequence Genome evolution Negative selection: the removal of deleterious mutations from a population (purifying selection) Positive selection: the retention of mutations that benefit an organism Genome function Functional sequences are subject to evolutionary selection, which can leave a signature in the aligned sequences neutral advantageous disadvantageous 6

7 Homolog, ortholog, paralog Contemporary org Speciation Speciation Intermediate org Gene duplication Homolog: two sequences that share a common evolutionary ancestry Ortholog: homologous sequences in different species that arose from a common ancestral gene during speciation. Orthologs often, but not always, have the same function. Paralog: homologous sequences that arose by gene duplication. Paralogous sequences provide useful insight to the way genomes evolve. Ancestral org 7

8 Detecting orthologous genes Best reciprocal Blast hit Lose of FOXJ2 in the fish lineage g1_a g1_b g1_a g1_b FOXJ2 g2_a g2_b g2_a g2_b Phylogenetic tree of the gene family Combine reciprocal best matches with surrounding genomic information, e.g. neighbor genes FOXJ3 FOXJ3 duplication in the fish lineage 8 Hubbard et al. NAR 35:D610, 2007

9 Synteny conservation Synteny: the occurrence of two or more gene loci on the same chromosome, regardless of whether or not they are genetically linked. Conserved synteny: preserved co-localization of genes on chromosomes of related species. Mouse Genome Sequencing Consortium. Nature 420:520,

10 Conserved Synteny Human Chr1 Chimpanzee (5 million years) Mouse (75-80 million years) Human-mouse: Conserved synteny at a higher degree than expected, with very little micro-rearrangement, and up to 95% conserved synteny. Human-fly: limited if any conserved syntenic stretches, possibly only those due to functional constraints, e.g. Hox cluster. 10

11 COG: Classify proteins encoded in complete genomes on the basis of the orthology concept COG (Clusters of Orthologous Groups of proteins): prokaryotes and unicellular eukaryotes KOG: eukaryotic orthologous groups Allows one to transfer functional information from experimentally characterized proteins to their orthologs from poorly studied organisms 11

12 KEGG: TCA cycle, E. coli TCA cycle, yeast TCA cycle, reference pathway TCA cycle, Dog 12

13 Evolution of orthologs: Ka/Ks Align the cdna sequences in accordance with their pairwise amino acid alignments Synonymous mutation: mutations that do not cause an amino acid replacement, e.g. AGG (Arginine) -> AGA (Arginine) Nonsynonymous mutation: mutations that result in amino acid replacements, e.g. AGG (Arginine) -> AGC (Serine) Ka/Ks ratio: Ratio between the rates of nonsynonymous mutations and synonymous mutations Indicator of selective pressure acting on a protein-coding gene Ortholog pairs generally have low values of Ka/Ks (<0.05) Proteins with Ka/Ks>1 are usually defined as being subject to positive selection 13

14 Positive selection Human-chimp comparison, positive selection Immune response Human-chimp-mouse comparison, positive selection in human lineage (Clark et al. Science 302:1960, 2003) Null hypothesis: all three branches have two classes of amino acid residues, those are neutrally evolving (Ka/Ks=1) and those that are under negative selection (Ka/Ks<1) Alternative hypothesis: human lineage have a subset of sites with accelerated amino acid substitution (Ka/Ks>1) 1,547 genes: Neurogenesis, Speech etc. 14

15 Proteins are not enough to explain the diversity Human-mouse (Mouse Genome Sequencing Consortium. Nature 420:520, 2002) 99% of the protein coding genes in mouse align with homologs in human 80% of the protein coding mouse genes have clear 1:1 human orthologs Yeast-worm-fly (Rubin et al. Science 287:2204, 2000) Non-redundant protein sets Yeast: 4,383 Worm: 9,453 Fly: 8,065 15

16 Whole genome alignment Needleman and Wunsch and Smith-Waterman are not appropriate Tools Can t handle rearranged sequences Time and memory usage expensive Local aligners: BLASTZ, BLAT Global aligners: AVID, LAGAN Three steps Seeding Extension/anchoring Gapped alignment Ureta-Vidal et al. Nat Rev Genet 4:251,

17 Pre-computed alignments UCSC genome browser: Ensembl: Phenylalanine hydroxylase (PAH): deficiency of this enzyme activity results in the autosomal recessive disorder phenylketonuria (PKU). 17 Miller et al. Genome Res 17:1797, 2007

18 Proportion of genome under purifying selection Not all the aligning DNA is functional, especially when the phylogenetic separation is not huge 40% of the human genome aligns with the mouse genome, including ancestral repeats (Presumed nonfunctional) Compare the quality of the alignments to those in the ancestral repeats (provides an estimation of the neutral rate of substitution) 5% of the human genome is under purifying selection: three times larger than the portion coding for proteins (Mouse Genome Sequencing Consortium. Nature 420:520, 2002) Sequences involved in regulation of gene expression Noncoding RNA 18

19 Composition of conserved sequence elements The proportion of conserved sequences not assigned to a transcript (CDS, 5 UTR, 3 UTR) is growing with increasing organism complexity. In human, nearly 70% of conserved element are not corresponding to any transcript, and therefore potentially control elements. Siepel et al. Genome Res. 15:1034,

20 Phylogenetic footprinting cis-regulatory elements: a region of DNA or RNA that regulates the expression of genes located on that same strand. Phylogenetic footprinting: An approach that seeks to identify conserved regulatory elements by comparing genomic sequences between related species. Decide on the gene of interest. Carefully choose species with orthologous genes. Decide on the length of the upstream or maybe downstream region to be looked at. Align the sequences. Look for conserved regions and analyze them. 20

21 Phylogenetic footprinting Decide on the gene of interest (SHH, sonic hedgehog: misexpression may lead to a common human limb malformation: preaxial polydactyly. ). Carefully choose species with orthologous genes (mouse-fugu). Decide on the length of the upstream or maybe downstream region to be looked at (may be very long). Align the sequences. Look for conserved regions and analyze them. Boffelli, et al. Nature Rev Genet 5:456,

22 Phylogenetic footprinting Four species of yeast (Kellis et al. Nature 423:241, 2003) Human, mouse, rat, dog (Xie et al. Nature 434:338, 2005) Kellis et al. Nature 423:241,

23 Non-coding RNAs trna, rrna micrornas (18-25nt): gene expression control Small RNAs: snornas, smrnas, pirnas, etc., cell machinery Long ncrnas (>200nt): Not very well known 23

24 Regulated expression of ultraconserved ncrnas HCC: hepatocellular carcinomas CRC: colorectal carcinomas CLL: leukemias Calin et al. Cancer Cell 12:215,

25 UCSC Genome Browser: _at 25

26 Summary Comparative genomics Genome evolution Genome function Ortholog vs paralog Conserved synteny Ka/Ks ratio and positive selection Whole genome alignment Conserved non-coding region Phylogenetic footprinting Non-coding RNA Hardison. PLoS Biol 1:156,