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

Abstract

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.

Qualifying the relationship between sequence conservation and molecular function

“…While such obstacles are more problematic when comparing highly divergent species, they are not restricted to comparisons in wide scopes. Genomic sequence alignment, a prerequisite to any constraint-based analysis, remains a challenging problem even for relatively closely related species Margulies et al 2007).…”

“…These studies include cell-based assays on a genomic scale (Kim et al 2005;Borneman et al 2007;The ENCODE Project Consortium 2007;Heintzman et al 2007;Xi et al 2007) and in vivo assays for developmentally important functions in individual loci in animal model organisms Brown et al 2007). Interestingly, they have demonstrated that there are large numbers of functional sequences that are not detectably conserved across both distant (McGaughey et al 2008, this issue) and close Margulies et al 2007) evolutionary timescales. This lack of conservation has several explanations in principle, each of which has distinct implications for functional annotation of complex genomes and a better understanding of genomic evolution.…”

Integration of Complex Data Sources to Provide Biologic Insight into Pulmonary Vascular Disease (2015 Grover Conference Series)

“…While useful in an earlier era, such oversimplification has limited our full understanding of complex human disease. Data have been accumulating from vast endeavors of high-throughput -omics profiling in general (e.g., the ENCODE project, [4][5][6][7] which has sought to identify all functional elements in the human genome). In this postgenomic era, we also appreciate that PH is driven by a complex and interconnected network of molecular processes, but these networks are only just beginning to be defined and catalogued in this disease.…”

618 Five vertebrate ChIP-seq reveals the evolutionary dynamics of transcription factor binding

“…Many comparative genomics approaches assume that a multi-species alignment of a high quality motif is indicative of functionality (19)(20)(21)(22)(23)(24)(25). Our analysis of experimentally determined in vivo occupancy of two TFs in multiple vertebrates revealed apparent limitations to this model and a number of other insights about the complex relationship between genetic sequence, transcription factor binding, and genome regulation.…”