The genetic structure of the indigenous hunter-gatherer peoples of southern Africa, the oldest known lineage of modern human, is important for understanding human diversity. Studies based on mitochondrial1 and small sets of nuclear markers2 have shown that these hunter-gatherers, known as Khoisan, San, or Bushmen, are genetically divergent from other humans1,3. However, until now, fully sequenced human genomes have been limited to recently diverged populations4–8. Here we present the complete genome sequences of an indigenous hunter-gatherer from the Kalahari Desert and a Bantu from southern Africa, as well as protein-coding regions from an additional three hunter-gatherers from disparate regions of the Kalahari. We characterize the extent of whole-genome and exome diversity among the five men, reporting 1.3 million novel DNA differences genome-wide, including 13,146 novel amino acid variants. In terms of nucleotide substitutions, the Bushmen seem to be, on average, more different from each other than, for example, a European and an Asian. Observed genomic differences between the hunter-gatherers and others may help to pinpoint genetic adaptations to an agricultural lifestyle. Adding the described variants to current databases will facilitate inclusion of southern Africans in medical research efforts, particularly when family and medical histories can be correlated with genome-wide data.

Background: The classical Bordetella subspecies are phylogenetically closely related, yet differ in some of the most interesting and important characteristics of pathogens, such as host range, virulence and persistence. The compelling picture from previous comparisons of the three sequenced genomes was of genome degradation, with substantial loss of genome content (up to 24%) associated with adaptation to humans. Results: For a more comprehensive picture of lineage evolution, we employed comparative genomic and phylogenomic analyses using seven additional diverse, newly sequenced Bordetella isolates. Genome-wide single nucleotide polymorphism (SNP) analysis supports a reevaluation of the phylogenetic relationships between the classical Bordetella subspecies, and suggests a closer link between ovine and human B. parapertussis lineages than has been previously proposed. Comparative analyses of genome content revealed that only 50% of the pan-genome is conserved in all strains, reflecting substantial diversity of genome content in these closely related pathogens that may relate to their different host ranges, virulence and persistence characteristics. Strikingly, these analyses suggest possible horizontal gene transfer (HGT) events in multiple loci encoding virulence factors, including O-antigen and pertussis toxin (Ptx). Segments of the pertussis toxin locus (ptx) and its secretion system locus (ptl) appear to have been acquired by the classical Bordetella subspecies and are divergent in different lineages, suggesting functional divergence in the classical Bordetellae.

Despite the fact that closely related bacteria can cause different levels of disease, the genetic changes that cause some isolates to be more pathogenic than others are generally not well understood. We use a combination of approaches to determine which factors contribute to the increased virulence of a Bordetella bronchiseptica lineage. A strain isolated from a host with B. bronchiseptica-induced disease, strain 1289, was 60-fold more virulent in mice than one isolated from an asymptomatically infected host, strain RB50. Transcriptome analysis and quantitative reverse transcription-PCR showed that the type III secretion system (TTSS) genes were more highly expressed by strain 1289 than strain RB50. Compared to strain RB50, strain 1289 exhibited greater TTSS-mediated cytotoxicity of a mammalian cell line. Additionally, we show that the increase in virulence of strain 1289 compared to that of RB50 was partially attributable to the TTSS. Using multilocus sequence typing, we identified another strain from the same lineage as strain 1289. Similar to strain 1289, we implicate the TTSS in the increased virulence of this strain. Together, our data suggest that the TTSS is involved in the increased virulence of a B. bronchiseptica lineage which appears to be disproportionately associated with disease. These data are consistent with the view that B. bronchiseptica lineages can have different levels of virulence, which may contribute to this species' ability to cause different severities of respiratory disease.

Bordetella bronchiseptica is a gram-negative respiratory pathogen that infects a wide range of hosts and causes a diverse spectrum of disease. This diversity is likely affected by multiple factors, such as host immune status, polymicrobial infection, and strain diversity. In a murine model of infection, we found that the virulence of B. bronchiseptica strains, as measured by the mean lethal dose, varied widely. Strain 253 was less virulent than the typically studied strain, RB50. Transcriptome analysis showed that cyaA, the gene encoding adenylate cyclase toxin (CyaA), was the most downregulated transcript identified in strain 253 compared to that in strain RB50. Comparative genomic hybridization and genome sequencing of strain 253 revealed that the cya locus, which encodes, activates, and secretes CyaA, was replaced by an operon (ptp) predicted to encode peptide transport proteins. Other B. bronchiseptica strains from the same phylogenetic lineage as that of strain 253 also lacked the cya locus, contained the ptp genes, and were less virulent than strain RB50. Although the loss of CyaA would be expected to be counterselected since it is conserved among the classical bordetellae and believed to be important to their success, our data indicate that the loss of this toxin and the gain of the ptp genes occurred in an ancestral strain that then expanded into a lineage. This suggests that there may be ecological niches in which CyaA is not critical for the success of B. bronchiseptica.

Summary: We develop a novel mining pipeline, Integrative Next-generation Genome Analysis Pipeline (inGAP), guided by a Bayesian principle to detect single nucleotide polymorphisms (SNPs), insertion/deletions (indels) by comparing high-throughput pyrosequencing reads with a reference genome of related organisms. inGAP can be applied to the mapping of both Roche/454 and Illumina reads with no restriction of read length. Experiments on simulated and experimental data show that this pipeline can achieve overall 97% accuracy in SNP detection and 94% in the finding of indels. All the detected SNPs/indels can be further evaluated by a graphical editor in our pipeline. inGAP also provides functions of multiple genomes comparison and assistance of bacterial genome assembly.Availability: inGAP is available at http://sites.google.com/site/nextgengenomics/ingapContact: scs@bx.psu.eduSupplementary information: Supplementary data are available at Bioinformatics online.