Article

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

Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Genome Research (Impact Factor: 13.85). 07/2007; 17(6):760-74. DOI: 10.1101/gr.6034307
Source: PubMed

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.

0 Followers
 · 
378 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In addition to their protein coding function, exons can also serve as transcriptional enhancers. Mutations in these exonic-enhancers (eExons) could alter both protein function and transcription. However, the functional consequence of eExon mutations is not well known. Here, using massively parallel reporter assays, we dissect the enhancer activity of three liver eExons (SORL1 exon 17, TRAF3IP2 exon 2, PPARG exon 6) at single nucleotide resolution in the mouse liver. We find that both synonymous and non-synonymous mutations have similar effects on enhancer activity and many of the deleterious mutation clusters overlap known liver-associated transcription factor binding sites. Carrying a similar massively parallel reporter assay in HeLa cells with these three eExons found differences in their mutation profiles compared to the liver, suggesting that enhancers could have distinct operating profiles in different tissues. Our results demonstrate that eExon mutations could lead to multiple phenotypes by disrupting both the protein sequence and enhancer activity and that enhancers can have distinct mutation profiles in different cell types.
    PLoS Genetics 10/2014; 10(10):e1004592. DOI:10.1371/journal.pgen.1004592 · 8.17 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The 3' UTRs of eukaryotic genes participate in a variety of post-transcriptional (and some transcriptional) regulatory interactions. Some of these interactions are well characterised, but an undetermined number remain to be discovered. While some regulatory sequences in 3' UTRs may be conserved over long evolutionary time scales, others may have only ephemeral functional significance as regulatory profiles respond to changing selective pressures. Here we propose a sensitive segmentation methodology for investigating patterns of composition and conservation in 3' UTRs based on comparison of closely related species. We describe encodings of pairwise and three-way alignments integrating information about conservation, GC content and transition/transversion ratios and apply the method to three closely related Drosophila species: D. melanogaster, D. simulans and D. yakuba. Incorporating multiple data types greatly increased the number of segment classes identified compared to similar methods based on conservation or GC content alone. We propose that the number of segments and number of types of segment identified by the method can be used as proxies for functional complexity. Our main finding is that the number of segments and segment classes identified in 3' UTRs is greater than in the same length of protein-coding sequence, suggesting greater functional complexity in 3' UTRs. There is thus a need for sustained and extensive efforts by bioinformaticians to delineate functional elements in this important genomic fraction. C code, data and results are available upon request.
    PLoS ONE 05/2014; 9(5):e97336. DOI:10.1371/journal.pone.0097336 · 3.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Multiple sequence alignments (MSAs) are a prerequisite for a wide variety of evolutionary analyses. Published assessments and benchmark datasets for protein and, to a lesser extent, global nucleotide MSAs are available, but less effort has been made to establish benchmarks in the more general problem of whole genome alignment (WGA). Using the same model as the successful Assemblathon competitions we organized a competitive evaluation in which teams submitted their alignments and then assessments were performed collectively after all the submissions were received. Three datasets were used; two were simulated and based on primate and mammalian phylogenies and one was comprised of 20 real fly genomes. In total 35 submissions were assessed, submitted by ten teams using 12 different alignment pipelines. We found agreement between independent simulation-based and statistical assessments indicating that there are substantial accuracy differences between contemporary alignment tools. We saw considerable difference in the alignment quality of differently annotated regions and found few tools aligned the duplications analysed. We found many tools worked well at shorter evolutionary distances, but fewer performed competitively at longer distances. We provide all datasets, submissions and assessment programs for further study and provide, as a resource for future benchmarking, a convenient repository of code and data for reproducing the simulation assessments.
    Genome Research 10/2014; 24(12). DOI:10.1101/gr.174920.114 · 13.85 Impact Factor

Full-text (2 Sources)

Download
99 Downloads
Available from
May 21, 2014