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.

Comparative sequencing provides insights about the structure and conservation of marsupial and monotreme genomes.

Sequencing and comparative analyses of genomes from multiple vertebrates are providing insights about the genetic basis for biological diversity. To date, these efforts largely have focused on eutherian mammals, chicken, and fish. In this article, we describe the generation and study of genomic sequences from noneutherian mammals, a group of species occupying unusual phylogenetic positions. A large sequence data set (totaling >5 Mb) was generated for the same orthologous region in three marsupial (North American opossum, South American opossum, and Australian tammar wallaby) and one monotreme (platypus) genomes. These ancient mammalian genomes are characterized by unusual architectural features with respect to G + C and repeat content, as well as compression relative to human. Approximately 14% and 34% of the human sequence forms alignments with the orthologous sequence from platypus and the marsupials, respectively; these numbers are distinctly lower than that observed with nonprimate eutherian mammals (45-70%). The alignable sequences between human and each marsupial species are not completely overlapping (only 80% common to all three species) nor are the platypus-alignable sequences completely contained within the marsupial-alignable sequences. Phylogenetic analysis of synonymous coding positions reveals that platypus has a notably long branch length, with the human-platypus substitution rate being on average 55% greater than that seen with human-marsupial pairs. Finally, analyses of the major mammalian lineages reveal distinct patterns with respect to the common presence of evolutionarily conserved vertebrate sequences. Our results confirm that genomic sequence from noneutherian mammals can contribute uniquely to unraveling the functional and evolutionary histories of the mammalian genome.

Parallel construction of orthologous sequence-ready clone contig maps in multiple species.

Comparison is a fundamental tool for analyzing DNA sequence. Interspecies sequence comparison is particularly powerful for inferring genome function and is based on the simple premise that conserved sequences are likely to be important. Thus, the comparison of a genomic sequence with its orthologous counterpart from another species is increasingly becoming an integral component of genome analysis. In ideal situations, such comparisons are performed with orthologous sequences from multiple species. To facilitate multispecies comparative sequence analysis, a robust and scalable strategy for simultaneously constructing sequence-ready bacterial artificial chromosome (BAC) contig maps from targeted genomic regions has been developed. Central to this approach is the generation and utilization of "universal" oligonucleotide-based hybridization probes ("overgo" probes), which are designed from sequences that are highly conserved between distantly related species. Large collections of these probes are used en masse to screen BAC libraries from multiple species in parallel, with the isolated clones assembled into physical contig maps. To validate the effectiveness of this strategy, efforts were focused on the construction of BAC-based physical maps from multiple mammalian species (chimpanzee, baboon, cat, dog, cow, and pig). Using available human and mouse genomic sequence and a newly developed computer program to design the requisite probes, sequence-ready maps were constructed in all species for a series of targeted regions totaling approximately 16 Mb in the human genome. The described approach can be used to facilitate the multispecies comparative sequencing of targeted genomic regions and can be adapted for constructing BAC contig maps in other vertebrates.

Pericentromeric duplications in the laboratory mouse.

Duplications have long been postulated to be an important mechanism by which genomes evolve. Interspecies genomic comparisons are one method by which the origin and molecular mechanism of duplications can be inferred. By comparative mapping in human, mouse, and rat, we previously found evidence for a recent chromosome-fission event that occurred in the mouse lineage. Cytogenetic mapping revealed that the genomic segments flanking the fission site appeared to be duplicated, with copies residing near the centromere of multiple mouse chromosomes. Here we report the mapping and sequencing of the regions of mouse chromosomes 5 and 6 involved in this chromosome-fission event as well as the results of comparative sequence analysis with the orthologous human and rat genomic regions. Our data indicate that the duplications associated with mouse chromosomes 5 and 6 are recent and that the resulting duplicated segments share significant sequence similarity with a series of regions near the centromeres of the mouse chromosomes previously identified by cytogenetic mapping. We also identified pericentromeric duplicated segments shared between mouse chromosomes 5 and 1. Finally, novel mouse satellite sequences as well as putative chimeric transcripts were found to be associated with the duplicated segments. Together, these findings demonstrate that pericentromeric duplications are not restricted to primates and may be a common mechanism for genome evolution in mammals.