The ongoing generation of prodigious amounts of genomic sequence data from myriad vertebrates is providing unparalleled opportunities for establishing definitive phylogenetic relationships among species. The size and complexities of such comparative sequence data sets not only allow smaller and more difficult branches to be resolved but also present unique challenges, including large computational requirements and the negative consequences of systematic biases. To explore these issues and to clarify the phylogenetic relationships among mammals, we have analyzed a large data set of over 60 megabase pairs (Mb) of high-quality genomic sequence, which we generated from 41 mammals and 3 other vertebrates. All sequences are orthologous to a 1.9-Mb region of the human genome that encompasses the cystic fibrosis transmembrane conductance regulator gene (CFTR). To understand the characteristics and challenges associated with phylogenetic analyses of such a large data set, we partitioned the sequence data in several ways and utilized maximum likelihood, maximum parsimony, and Neighbor-Joining algorithms, implemented in parallel on Linux clusters. These studies yielded well-supported phylogenetic trees, largely confirming other recent molecular phylogenetic analyses. Our results provide support for rooting the placental mammal tree between Atlantogenata (Xenarthra and Afrotheria) and Boreoeutheria (Euarchontoglires and Laurasiatheria), illustrate the difficulty in resolving some branches even with large amounts of data (e.g., in the case of Laurasiatheria), and demonstrate the valuable role that very large comparative sequence data sets can play in refining our understanding of the evolutionary relationships of vertebrates.

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 (soop) to design the requisite probes, sequence-ready maps were constructed in all species for a series of targeted regions totaling ∼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.

The sharing of genome sequences in online data repositories allows for large scale analyses of specific genes or gene families. This can result in the detection of novel gene subtypes as well as the development of improved detection methods. Here, we used publicly available WGS data to detect a novel Stx subtype, Stx2n in two clinical E. coli strains isolated in the USA. During this process, additional Stx2 subtypes were detected; six Stx2j, one Stx2m strain, and one Stx2o, were all analyzed for variability from the originally described subtypes. Complete genome sequences were assembled from short- or long-read sequencing and analyzed for serotype, and ST types. The WGS data from Stx2n- and Stx2o-producing STEC strains were further analyzed for virulence genes pro-phage analysis and phage insertion sites. Nucleotide and amino acid maximum parsimony trees showed expected clustering of the previously described subtypes and a clear separation of the novel Stx2n subtype. WGS data were used to design OMNI PCR primers for the detection of all known stx1 (283 bp amplicon), stx2 (400 bp amplicon), intimin encoded by eae (221 bp amplicon), and stx2f (438 bp amplicon) subtypes. These primers were tested in three different laboratories, using standard reference strains. An analysis of the complete genome sequence showed variability in serogroup, virulence genes, and ST type, and Stx2 pro-phages showed variability in size, gene composition, and phage insertion sites. The strains with Stx2j, Stx2m, Stx2n, and Stx2o showed toxicity to Vero cells. Stx2j carrying strain, 2012C-4221, was induced when grown with sub-inhibitory concentrations of ciprofloxacin, and toxicity was detected. Taken together, these data highlight the need to reinforce genomic surveillance to identify the emergence of potential new Stx2 or Stx1 variants. The importance of this surveillance has a paramount impact on public health. Per our description in this study, we suggest that 2017C-4317 be designated as the Stx2n type-strain.

The transcription factor SOX10 is essential for all stages of Schwann cell development including myelination. SOX10 cooperates with other transcription factors to activate the expression of key myelin genes in Schwann cells and is therefore a context-dependent, pro-myelination transcription factor. As such, the identification of genes regulated by SOX10 will provide insight into Schwann cell biology and related diseases. While genome-wide studies have successfully revealed SOX10 target genes, these efforts mainly focused on myelinating stages of Schwann cell development. We propose that less-biased approaches will reveal novel functions of SOX10 outside of myelination. We developed a stringent, computational-based screen for genome-wide identification of SOX10 response elements. Experimental validation of a pilot set of predicted binding sites in multiple systems revealed that SOX10 directly regulates a previously unreported alternative promoter at SOX6, which encodes a transcription factor that inhibits glial cell differentiation. We further explored the utility of our computational approach by combining it with DNase-seq analysis in cultured Schwann cells and previously published SOX10 ChIP-seq data from rat sciatic nerve. Remarkably, this analysis enriched for genomic segments that map to loci involved in the negative regulation of gliogenesis including SOX5, SOX6, NOTCH1, HMGA2, HES1, MYCN, ID4, and ID2. Functional studies in Schwann cells revealed that: (1) all eight loci are expressed prior to myelination and down-regulated subsequent to myelination; (2) seven of the eight loci harbor validated SOX10 binding sites; and (3) seven of the eight loci are down-regulated upon repressing SOX10 function. Our computational strategy revealed a putative novel function for SOX10 in Schwann cells, which suggests a model where SOX10 activates the expression of genes that inhibit myelination during non-myelinating stages of Schwann cell development. Importantly, the computational and functional datasets we present here will be valuable for the study of transcriptional regulation, SOX protein function, and glial cell biology.