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Analyses of deep mammalian sequence alignments and constraint predictions for 1% of the human genome

A key component of the ongoing ENCODE project involves rigorous comparative sequence analyses for the initially targeted 1% of the human genome. Here, we present orthologous sequence generation, alignment, and evolutionary constraint analyses of 23 mammalian species for all ENCODE targets. Alignments were generated using four different methods; comparisons of these methods reveal large-scale consistency but substantial differences in terms of small genomic rearrangements, sensitivity (sequence coverage), and specificity (alignment accuracy). We describe the quantitative and qualitative trade-offs concomitant with alignment method choice and the levels of technical error that need to be accounted for in applications that require multisequence alignments. Using the generated alignments, we identified constrained regions using three different methods. While the different constraint-detecting methods are in general agreement, there are important discrepancies relating to both the underlying alignments and the specific algorithms. However, by integrating the results across the alignments and constraint-detecting methods, we produced constraint annotations that were found to be robust based on multiple independent measures. Analyses of these annotations illustrate that most classes of experimentally annotated functional elements are enriched for constrained sequences; however, large portions of each class (with the exception of protein-coding sequences) do not overlap constrained regions. The latter elements might not be under primary sequence constraint, might not be constrained across all mammals, or might have expendable molecular functions. Conversely, 40% of the constrained sequences do not overlap any of the functional elements that have been experimentally identified. Together, these findings demonstrate and quantify how many genomic functional elements await basic molecular characterization.

³ Present addresses:

Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA 98195, USA

⁴ Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison, Madison, WI 53706, USA

⁵ Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA 98195, USA

⁶ Department of Biological Statistics and Computational Biology, Cornell University, Ithaca, NY 14853, USA

⁷ Corresponding author.

E-mail elliott{at}nhgri.nih.gov; fax (301) 480-3520.

⁸ Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

⁹ Department of Genetics, Stanford University, Stanford, CA 94305, USA.

¹⁰ Department of Computer Science, Stanford University, Stanford, CA 94305, USA.

¹¹ Department of Biomolecular Engineering, University of California, Santa Cruz, CA 95064, USA.

¹² Center for Biomolecular Science and Engineering, University of California, Santa Cruz, CA 95064, USA.

¹³ Department of Electrical Engineering and Computer Sciences, University of California, Berkeley, CA 94720, USA.

¹⁴ European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton CB10 1SA, UK.

¹⁵ Department of Computer Science and Engineering, The Pennsylvania State University, University Park, PA 16802, USA.

¹⁶ Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland.

¹⁷ Department of Zoology and Animal Biology, Faculty of Sciences, University of Geneva, Geneva, Switzerland.

¹⁸ Department of Applied Science & Technology, University of California, Berkeley, CA 94720, USA.

¹⁹ Department of Statistics, University of California, Berkeley, CA 94720, USA.

²⁰ Department of Bioengineering, University of California, Berkeley, CA 94720-1762, USA.

²¹ NIH Intramural Sequencing Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

²² Center for Comparative Genomics and Bioinformatics, Huck Institutes for Life Sciences, Penn State University, University Park, PA 16802, USA.

²³ Howard Hughes Medical Institute, University of California, Santa Cruz, CA 95064, USA.

²⁴ Department of Mathematics, University of California, Berkeley, CA 94720, USA.

²⁵ Department of Pathology, Stanford University, Stanford, CA 94305, USA.

²⁶ Human Genome Sequencing Center and Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

²⁷ Genome Sequencing Center, Washington University School of Medicine, Campus Box 8501, 4444 Forest Park Avenue, St. Louis, MO 63108, USA.

²⁸ Broad Institute of Harvard and MIT, 320 Charles Street, Cambridge, MA 02141, USA.

²⁹ Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA.

³⁰ Canada’s Michael Smith Genome Sciences Centre, BC Cancer Research Centre, BC Cancer Agency, Vancouver, British Columbia V5Z 4S6, Canada.

[Supplemental material is available online at www.genome.org.]

Article is online at http://www.genome.org/cgi/doi/10.1101/gr.6034307

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