Analyses of deep mammalian sequence alignments and constraint predictions for 1% of the human genome
Authors: Margulies Elliott H. , Cooper Gregory M. , Asimenos George , Thomas Daryl J. , Dewey Colin N. , Siepel Adam , Birney Ewan , Keefe Damian , Schwartz Ariel S. , Hou Minmei , Taylor James , Nikolaev Sergey , Montoya-Burgos Juan I. , Löytynoja Ari , Whelan Simon , Pardi Fabio , Massingham Tim , Brown James B. , Bickel Peter , Holmes Ian , Mullikin James C. , Ureta-Vidal Abel , Paten Benedict , Stone Eric A. , Rosenbloom Kate R. , Kent W. James , Bouffard Gerard G. , Guan Xiaobin , Hansen Nancy F. , Idol Jacquelyn R. , Maduro Valerie V.B. , Maskeri Baishali , McDowell Jennifer C. , Park Morgan , Thomas Pamela J. , Young Alice C. , Blakesley Robert W. , Muzny Donna M. , Sodergren Erica , Wheeler David A. , Worley Kim C. , Jiang Huaiyang , Weinstock George M. , Gibbs Richard A. , Graves Tina , Fulton Robert , Mardis Elaine R. , Wilson Richard K. , Clamp Michele , Cuff James , Gnerre Sante , Jaffe David B. , Chang Jean L. , Lindblad-Toh Kerstin , Lander Eric S. , Hinrichs Angie , Trumbower Heather , Clawson Hiram , Zweig Ann , Kuhn Robert M. , Barber Galt , Harte Rachel , Karolchik Donna , Field Matthew A. , Moore Richard A. , Matthewson Carrie A. , Schein Jacqueline E. , Marra Marco A. , Antonarakis Stylianos E. , Batzoglou Serafim , Goldman Nick , Hardison Ross , Haussler David , Miller Webb , Pachter Lior , Green Eric D. , Sidow Arend 
Published: June 2007

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.