Comparative genomics of 29 eutherian mammals Kerstin Lindblad-Toh1,2, Manuel Garber1*, Or Zuk1*, Michael F. Lin1,3*, Brian J. Parker4*, Stefan Washietl3*, Pouya Kheradpour1,3*, Jason Ernst1,3*, Gregory Jordan5*, Evan Mauceli1*, Lucas D. Ward1,3*, Craig B. Lowe6,7,8*, Alisha Holloway9*, Michele Clamp1,10*, Sante Gnerre1*, Broad Institute Sequencing Platform Whole Genome Assembly Team, Kim C. Worley14, Christie L. Kovar14, Donna M. Muzny14, Richard A. Gibbs14, Baylor College of Medicine Human Genome Sequencing Center, Wesley C. Warren15, Elaine R Mardis15, George M. Weinstock14,15, Richard K. Wilson15, Washington University Genome Center, Ewan Birney5, Elliott Margulies16, Javier Herrero5, Eric Green17, David Haussler6,8, Adam Siepel12, Nick Goldman5, Katherine S. Pollard9,18, Jakob S. Pedersen4,19, Eric S. Lander1, Manolis Kellis1,3 Author Affiliations: 1 Broad Institute of Harvard and Massachusetts Institute of Technology (MIT), 2 Science for Life Laboratory, Uppsala University. 3 MIT Computer Science and Artificial Intelligence Laboratory. 4 The Bioinformatics Centre, Department of Biology, University of Copenhagen. 5 EMBL-EBI, Wellcome Trust Genome Campus. 6 Center for Biomolecular Science and Engineering, University of California. 7 Department of Developmental Biology, Stanford University. 8 Howard Hughes Medical Institute. 9 Gladstone Institutes, University of California. 10 BioTeam Inc. 14 Human Genome Sequencing Center, Baylor College of Medicine. 15 Washington University School of Medicine in Saint Louis. 16 Genome Informatics Section, National Human Genome Research Institute. 17 NISC Comparative Sequencing Program, NHGRI. 18 Institute for Human Genetics, and Division of Biostatistics, UC San Francisco. 19 Department of Molecular Medicine (MOMA), Aarhus University Hospital. Funding: This work was supported by the National Human Genome Research Institute (NHGRI), including grant U54 HG003273 (R.A.G), National Institute for General Medicine (NIGMS) grant #GM82901 (Pollard lab) and the European Science Foundation (EURYI award to K.L-T.), NSF National Science Foundation (NSF) postdoctoral fellowship award 0905968 (J.E.), NSF CAREER 0644282 and NIH R01 HG004037 and the Sloan Foundation (M.K.), and an Erwin Schrödinger Fellowship of the Austrian Fonds zur Förderung der Wissenschaftlichen Forschung (SW), the Gates Cambridge Trust (GJ), Novo Nordisk Foundation (BJP and JW); a Statistics Network Fellowship, Department of Mathematical Sciences, University of Copenhagen (BJP); the David and Lucile Packard Foundation (AS); the Danish Council for Independent Research | Medical Sciences (JSP); Lundbeck Foundation (JSP). Nucleotide-level measures of evolut. constraint Sequencing strategy for human genome annotation Evolutionary signatures for genome annotation Mammalian constraint matches Human SNPs Protein-coding genes - Codon Substitution Frequencies - Reading Frame Conservation Broad, Baylor, WashU RNA structures - Compensatory changes - Silent G-U substitutions microRNAs - Shape of conservation profile - Structural features: loops, pairs - Relationship with 3’UTR motifs 4  29 mammals 1  4 substs / site 50  10 bp detection Regulatory motifs - Mutations preserve consensus - Increased Branch Length Score - Genome-wide conservation Position-specific constraint measures bias in subst. patterns Many closely related species. Maximize total branch length Protein-coding genes Coverage depth higher in functional regions Human SNPs match mammalian-wide twofold constraint rhombomere 4 expression (Lampe et al., NAR 2008) rhombomere 2 expr. (Tümpel PNAS 2008) RNA structures Evidence of selection against deletions in functional regions Genome-wide agreement in selection vs. polymorphisms Detection of evolutionarily constrained elements Position-specific constraint in human popul. Promoter elements TSS, Motif TSS, Bound TSS, Motif, π log-odds (12mers) π log-odds (50mers) ω (12mers) 29 mammals 7.1/1.5/4.6 6.8/1.8/4.1 5.7/ 1.1/3.8 5.7/1.8/3.0 (HMRD) Human Mouse Rat Dog 4.2/0.0/0.0 5.3/0.1/0.3 4.5/0.0/0.0 5.1/0.6/1.7 Regulatory motifs Reduced heterozygocity evidence of human selection Even in absence of species-level conservation Estimated / kmers detectable at 5% FDR / base pairs detectable at 5% FDR Codon-specific measures of positive selection Chromatin states shed light on intergenic functions Gene-wide vs. punctate regions of exons positive selection