Comparative Analysis of Genome of Ehrlichia sp. HF, a Model Bacterium to Study Fatal Human Ehrlichiosis.

BACKGROUND: The genus Ehrlichia consists of tick-borne obligatory intracellular bacteria that can cause deadly diseases of medical and agricultural importance. Ehrlichia sp. HF, isolated from Ixodes ovatus ticks in Japan [also referred to as I. ovatus Ehrlichia (IOE) agent], causes acute fatal infection in laboratory mice that resembles acute fatal human monocytic ehrlichiosis caused by Ehrlichia chaffeensis. As there is no small laboratory animal model to study fatal human ehrlichiosis, Ehrlichia sp. HF provides a needed disease model. However, the inability to culture Ehrlichia sp. HF and the lack of genomic information have been a barrier to advance this animal model. In addition, Ehrlichia sp. HF has several designations in the literature as it lacks a taxonomically recognized name. RESULTS: We stably cultured Ehrlichia sp. HF in canine histiocytic leukemia DH82 cells from the HF strain-infected mice, and determined its complete genome sequence. Ehrlichia sp. HF has a single double-stranded circular chromosome of 1,148,904 bp, which encodes 866 proteins with a similar metabolic potential as E. chaffeensis. Ehrlichia sp. HF encodes homologs of all virulence factors identified in E. chaffeensis, including 23 paralogs of P28/OMP-1 family outer membrane proteins, type IV secretion system apparatus and effector proteins, two-component systems, ankyrin-repeat proteins, and tandem repeat proteins. Ehrlichia sp. HF is a novel species in the genus Ehrlichia, as demonstrated through whole genome comparisons with six representative Ehrlichia species, subspecies, and strains, using average nucleotide identity, digital DNA-DNA hybridization, and core genome alignment sequence identity. CONCLUSIONS: The genome of Ehrlichia sp. HF encodes all known virulence factors found in E. chaffeensis, substantiating it as a model Ehrlichia species to study fatal human ehrlichiosis. Comparisons between Ehrlichia sp. HF and E. chaffeensis will enable identification of in vivo virulence factors that are related to host specificity, disease severity, and host inflammatory responses. We propose to name Ehrlichia sp. HF as Ehrlichia japonica sp. nov. (type strain HF), to denote the geographic region where this bacterium was initially isolated.

Ancestral characterization of 1018 cancer cell lines highlights disparities and reveals gene expression and mutational differences.

BACKGROUND: Although cell lines are an essential resource for studying cancer biology, many are of unknown ancestral origin, and their use may not be optimal for evaluating the biology of all patient populations. METHODS: An admixture analysis was performed using genome-wide chip data from the Catalogue of Somatic Mutations in Cancer (COSMIC) Cell Lines Project to calculate genetic ancestry estimates for 1018 cancer cell lines. After stratifying the analyses by tissue and histology types, linear models were used to evaluate the influence of ancestry on gene expression and somatic mutation frequency. RESULTS: For the 701 cell lines with unreported ancestry, 215 were of East Asian origin, 30 were of African or African American origin, and 453 were of European origin. Notable imbalances were observed in ancestral representation across tissue type, with the majority of analyzed tissue types having few cell lines of African American ancestral origin, and with Hispanic and South Asian ancestry being almost entirely absent across all cell lines. In evaluating gene expression across these cell lines, expression levels of the genes neurobeachin line 1 (NBEAL1), solute carrier family 6 member 19 (SLC6A19), HEAT repeat containing 6 (HEATR6), and epithelial cell transforming 2 like (ECT2L) were associated with ancestry. Significant differences were also observed in the proportions of somatic mutation types across cell lines with varying ancestral proportions. CONCLUSIONS: By estimating genetic ancestry for 1018 cancer cell lines, the authors have produced a resource that cancer researchers can use to ensure that their cell lines are ancestrally representative of the populations they intend to affect. Furthermore, the novel ancestry-specific signal identified underscores the importance of ancestral awareness when studying cancer.

Grafting or pruning in the animal tree: lateral gene transfer and gene loss?

BACKGROUND: Lateral gene transfer (LGT), also known as horizontal gene transfer, into multicellular eukaryotes with differentiated tissues, particularly gonads, continues to be met with skepticism by many prominent evolutionary and genomic biologists. A detailed examination of 26 animal genomes identified putative LGTs in invertebrate and vertebrate genomes, concluding that there are fewer predicted LGTs in vertebrates/chordates than invertebrates, but there is still evidence of LGT into chordates, including humans. More recently, a reanalysis of a subset of these putative LGTs into vertebrates concluded that there is not horizontal gene transfer in the human genome. One of the genes in dispute is an N-acyl-aromatic-L-amino acid amidohydrolase (ENSG00000132744), which encodes ACY3. This gene was initially identified as a putative bacteria-chordate LGT but was later debunked as it has a significant BLAST match to a more recently deposited genome of Saccoglossus kowalevskii, a flatworm, Metazoan, and hemichordate. RESULTS: Using BLAST searches, HMM searches, and phylogenetics to assess the evidence for LGT, gene loss, and rate variation in ACY3/ASPA homologues, the most parsimonious explanation for the distribution of ACY3/ASPA genes in eukaryotes involves both gene loss and bacteria-animal LGT, albeit LGT that occurred hundreds of millions of years ago prior to the divergence of gnathostomes. CONCLUSIONS: ACY3/ASPA is most likely a bacteria-animal LGT. LGTs at these time scales in the ancestors of humans are not unexpected given the many known, well-characterized, and adaptive LGTs from bacteria to insects and nematodes.

Lateral gene transfer between prokaryotes and eukaryotes.

Lateral gene transfer (LGT) is an all-encompassing term for the movement of DNA between diverse organisms. LGT is synonymous with horizontal gene transfer, and the terms are used interchangeably throughout the scientific literature. While LGT has been recognized within the bacteria domain of life for decades, inter-domain LGTs are being increasingly described. LGTs between bacteria and complex multicellular organisms are of interest because they challenge the long-held dogma that such transfers could only occur in closely-related, single-celled organisms. Scientists will continue to challenge our understanding of LGT as we sequence more, diverse organisms, as we sequence more endosymbiont-colonized arthropods, and as we continue to appreciate LGT events, both young and old.

Modeling the integration of bacterial rRNA fragments into the human cancer genome.

BACKGROUND: Cancer is a disease driven by the accumulation of genomic alterations, including the integration of exogenous DNA into the human somatic genome. We previously identified in silico evidence of DNA fragments from a Pseudomonas-like bacteria integrating into the 5'-UTR of four proto-oncogenes in stomach cancer sequencing data. The functional and biological consequences of these bacterial DNA integrations remain unknown. RESULTS: Modeling of these integrations suggests that the previously identified sequences cover most of the sequence flanking the junction between the bacterial and human DNA. Further examination of these reads reveals that these integrations are rich in guanine nucleotides and the integrated bacterial DNA may have complex transcript secondary structures. CONCLUSIONS: The models presented here lay the foundation for future experiments to test if bacterial DNA integrations alter the transcription of the human genes.

A review of bacteria-animal lateral gene transfer may inform our understanding of diseases like cancer.

Lateral gene transfer (LGT) from bacteria to animals occurs more frequently than was appreciated prior to the advent of genome sequencing. In 2007, LGT from bacterial Wolbachia endosymbionts was detected in ~33% of the sequenced arthropod genomes using a bioinformatic approach. Today, Wolbachia/host LGT is thought to be widespread and many other cases of bacteria-animal LGT have been described. In insects, LGT may be more frequently associated with endosymbionts that colonize germ cells and germ stem cells, like Wolbachia endosymbionts. We speculate that LGT may occur from bacteria to a wide variety of eukaryotes, but only becomes vertically inherited when it occurs in germ cells. As such, LGT may happen routinely in somatic cells but never become inherited or fixed in the population. Lack of inheritance of such mutations greatly decreases our ability to detect them. In this review, we propose that such noninherited bacterial DNA integration into chromosomes in human somatic cells could induce mutations leading to cancer or autoimmune diseases in a manner analogous to mobile elements and viral integrations.

Extensive duplication of the Wolbachia DNA in chromosome four of Drosophila ananassae.

BACKGROUND: Lateral gene transfer (LGT) from bacterial Wolbachia endosymbionts has been detected in ~20% of arthropod and nematode genome sequencing projects. Many of these transfers are large and contain a substantial part of the Wolbachia genome. RESULTS: Here, we re-sequenced three D. ananassae genomes from Asia and the Pacific that contain large LGTs from Wolbachia. We find that multiple copies of the Wolbachia genome are transferred to the Drosophila nuclear genome in all three lines. In the D. ananassae line from Indonesia, the copies of Wolbachia DNA in the nuclear genome are nearly identical in size and sequence yielding an even coverage of mapped reads over the Wolbachia genome. In contrast, the D. ananassae lines from Hawaii and India show an uneven coverage of mapped reads over the Wolbachia genome suggesting that different parts of these LGTs are present in different copy numbers. In the Hawaii line, we find that this LGT is underrepresented in third instar larvae indicative of being heterochromatic. Fluorescence in situ hybridization of mitotic chromosomes confirms that the LGT in the Hawaii line is heterochromatic and represents ~20% of the sequence on chromosome 4 (dot chromosome, Muller element F). CONCLUSIONS: This collection of related lines contain large lateral gene transfers composed of multiple Wolbachia genomes that constitute >2% of the D. ananassae genome (~5 Mbp) and partially explain the abnormally large size of chromosome 4 in D. ananassae.

Single molecule sequencing and genome assembly of a clinical specimen of Loa loa, the causative agent of loiasis.

BACKGROUND: More than 20% of the world's population is at risk for infection by filarial nematodes and >180 million people worldwide are already infected. Along with infection comes significant morbidity that has a socioeconomic impact. The eight filarial nematodes that infect humans are Wuchereria bancrofti, Brugia malayi, Brugia timori, Onchocerca volvulus, Loa loa, Mansonella perstans, Mansonella streptocerca, and Mansonella ozzardi, of which three have published draft genome sequences. Since all have humans as the definitive host, standard avenues of research that rely on culturing and genetics have often not been possible. Therefore, genome sequencing provides an important window into understanding the biology of these parasites. The need for large amounts of high quality genomic DNA from homozygous, inbred lines; the availability of only short sequence reads from next-generation sequencing platforms at a reasonable expense; and the lack of random large insert libraries has limited our ability to generate high quality genome sequences for these parasites. However, the Pacific Biosciences single molecule, real-time sequencing platform holds great promise in reducing input amounts and generating sufficiently long sequences that bypass the need for large insert paired libraries. RESULTS: Here, we report on efforts to generate a more complete genome assembly for L. loa using genetically heterogeneous DNA isolated from a single clinical sample and sequenced on the Pacific Biosciences platform. To obtain the best assembly, numerous assemblers and sequencing datasets were analyzed, combined, and compared. Quiver-informed trimming of an assembly of only Pacific Biosciences reads by HGAP2 was selected as the final assembly of 96.4 Mbp in 2,250 contigs. This results in ~9% more of the genome in ~85% fewer contigs from ~80% less starting material at a fraction of the cost of previous Roche 454-based sequencing efforts. CONCLUSIONS: The result is the most complete filarial nematode assembly produced thus far and demonstrates the utility of single molecule sequencing on the Pacific Biosciences platform for genetically heterogeneous metazoan genomes.

Rapid transcriptome sequencing of an invasive pest, the brown marmorated stink bug Halyomorpha halys.

BACKGROUND: Halyomorpha halys (Stål) (Insecta:Hemiptera;Pentatomidae), commonly known as the Brown Marmorated Stink Bug (BMSB), is an invasive pest of the mid-Atlantic region of the United States, causing economically important damage to a wide range of crops. Native to Asia, BMSB was first observed in Allentown, PA, USA, in 1996, and this pest is now well-established throughout the US mid-Atlantic region and beyond. In addition to the serious threat BMSB poses to agriculture, BMSB has become a nuisance to homeowners, invading home gardens and congregating in large numbers in human-made structures, including homes, to overwinter. Despite its significance as an agricultural pest with limited control options, only 100 bp of BMSB sequence data was available in public databases when this project began. RESULTS: Transcriptome sequencing was undertaken to provide a molecular resource to the research community to inform the development of pest control strategies and to provide molecular data for population genetics studies of BMSB. Using normalized, strand-specific libraries, we sequenced pools of all BMSB life stages on the Illumina HiSeq. Trinity was used to assemble 200,000 putative transcripts in >100,000 components. A novel bioinformatic method that analyzed the strand-specificity of the data reduced this to 53,071 putative transcripts from 18,573 components. By integrating multiple other data types, we narrowed this further to 13,211 representative transcripts. CONCLUSIONS: Bacterial endosymbiont genes were identified in this dataset, some of which have a copy number consistent with being lateral gene transfers between endosymbiont genomes and Hemiptera, including ankyrin-repeat related proteins, lysozyme, and mannanase. Such genes and endosymbionts may provide novel targets for BMSB-specific biocontrol. This study demonstrates the utility of strand-specific sequencing in generating shotgun transcriptomes and that rapid sequencing shotgun transcriptomes is possible without the need for extensive inbreeding to generate homozygous lines. Such sequencing can provide a rapid response to pest invasions similar to that already described for disease epidemiology.

Horizontal gene transfer between bacteria and animals.

Horizontal gene transfer is increasingly described between bacteria and animals. Such transfers that are vertically inherited have the potential to influence the evolution of animals. One classic example is the transfer of DNA from mitochondria and chloroplasts to the nucleus after the acquisition of these organelles by eukaryotes. Even today, many of the described instances of bacteria-to-animal transfer occur as part of intimate relationships such as those of endosymbionts and their invertebrate hosts, particularly insects and nematodes, while numerous transfers are also found in asexual animals. Both of these observations are consistent with modern evolutionary theory, in particular the serial endosymbiotic theory and Muller's ratchet. Although it is tempting to suggest that these particular lifestyles promote horizontal gene transfer, it is difficult to ascertain given the nonrandom sampling of animal genome sequencing projects and the lack of a systematic analysis of animal genomes for such transfers.

Bacteria-human somatic cell lateral gene transfer is enriched in cancer samples.

There are 10× more bacterial cells in our bodies from the microbiome than human cells. Viral DNA is known to integrate in the human genome, but the integration of bacterial DNA has not been described. Using publicly available sequence data from the human genome project, the 1000 Genomes Project, and The Cancer Genome Atlas (TCGA), we examined bacterial DNA integration into the human somatic genome. Here we present evidence that bacterial DNA integrates into the human somatic genome through an RNA intermediate, and that such integrations are detected more frequently in (a) tumors than normal samples, (b) RNA than DNA samples, and (c) the mitochondrial genome than the nuclear genome. Hundreds of thousands of paired reads support random integration of Acinetobacter-like DNA in the human mitochondrial genome in acute myeloid leukemia samples. Numerous read pairs across multiple stomach adenocarcinoma samples support specific integration of Pseudomonas-like DNA in the 5'-UTR and 3'-UTR of four proto-oncogenes that are up-regulated in their transcription, consistent with conversion to an oncogene. These data support our hypothesis that bacterial integrations occur in the human somatic genome and may play a role in carcinogenesis. We anticipate that the application of our approach to additional cancer genome projects will lead to the more frequent detection of bacterial DNA integrations in tumors that are in close proximity to the human microbiome.

Neisseria meningitidis is structured in clades associated with restriction modification systems that modulate homologous recombination.

Molecular data on a limited number of chromosomal loci have shown that the population of Neisseria meningitidis (Nm), a deadly human pathogen, is structured in distinct lineages. Given that the Nm population undergoes substantial recombination, the mechanisms resulting in the evolution of these lineages, their persistence in time, and the implications for the pathogenicity of the bacterium are not yet completely understood. Based on whole-genome sequencing, we show that Nm is structured in phylogenetic clades. Through acquisition of specific genes and through insertions and rearrangements, each clade has acquired and remodeled specific genomic tracts, with the potential to impact on the commensal and virulence behavior of Nm. Despite this clear evidence of a structured population, we confirm high rates of detectable recombination throughout the whole Nm chromosome. However, gene conversion events were found to be longer within clades than between clades, suggesting a DNA cleavage mechanism associated with the phylogeny of the species. We identify 22 restriction modification systems, probably acquired by horizontal gene transfer from outside of the species/genus, whose distribution in the different strains coincides with the phylogenetic clade structure. We provide evidence that these clade-associated restriction modification systems generate a differential barrier to DNA exchange consistent with the observed population structure. These findings have general implications for the emergence of lineage structure and virulence in recombining bacterial populations, and they could provide an evolutionary framework for the population biology of a number of other bacterial species that show contradictory population structure and dynamics.

Improved metagenome assemblies through selective enrichment of bacterial genomic DNA from eukaryotic host genomic DNA using ATAC-seq.

Genomics can be used to study the complex relationships between hosts and their microbiota. Many bacteria cannot be cultured in the laboratory, making it difficult to obtain adequate amounts of bacterial DNA and to limit host DNA contamination for the construction of metagenome-assembled genomes (MAGs). For example, Wolbachia is a genus of exclusively obligate intracellular bacteria that live in a wide range of arthropods and some nematodes. While Wolbachia endosymbionts are frequently described as facultative reproductive parasites in arthropods, the bacteria are obligate mutualistic endosymbionts of filarial worms. Here, we achieve 50-fold enrichment of bacterial sequences using ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) with Brugia malayi nematodes, containing Wolbachia (wBm). ATAC-seq uses the Tn5 transposase to cut and attach Illumina sequencing adapters to accessible DNA lacking histones, typically thought to be open chromatin. Bacterial and mitochondrial DNA in the lysates are also cut preferentially since they lack histones, leading to the enrichment of these sequences. The benefits of this include minimal tissue input (<1 mg of tissue), a quick protocol (<4 h), low sequencing costs, less bias, correct assembly of lateral gene transfers and no prior sequence knowledge required. We assembled the wBm genome with as few as 1 million Illumina short paired-end reads with >97% coverage of the published genome, compared to only 12% coverage with the standard gDNA libraries. We found significant bacterial sequence enrichment that facilitated genome assembly in previously published ATAC-seq data sets from human cells infected with Mycobacterium tuberculosis and C. elegans contaminated with their food source, the OP50 strain of E. coli. These results demonstrate the feasibility and benefits of using ATAC-seq to easily obtain bacterial genomes to aid in symbiosis, infectious disease, and microbiome research.

Common analysis of direct RNA sequencinG CUrrently leads to misidentification of m5C at GCU motifs.

RNA modifications, such as methylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m5C) modifications is Tombo, which uses an "Alternative Model" to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including viruses, bacteria, fungi, and animals. The algorithm consistently identified a m5C at the central position of a GCU motif. However, it also identified a m5C in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this is a frequent false prediction. In the absence of further validation, several published predictions of m5C in a GCU context should be reconsidered, including those from human coronavirus and human cerebral organoid samples.

Extensively duplicated and transcriptionally active recent lateral gene transfer from a bacterial Wolbachia endosymbiont to its host filarial nematode Brugia malayi.

BACKGROUND: Lymphatic filariasis is a neglected tropical disease afflicting more than 120 million people, while another 1.3 billion people are at risk of infection. The nematode worm Brugia malayi is one of the causative agents of the disease and exists in a mutualistic symbiosis with Wolbachia bacteria. Since extensive lateral gene transfer occurs frequently between Wolbachia and its hosts, we sought to measure the extent of such LGT in B. malayi by whole genome sequencing of Wolbachia-depleted worms. RESULTS: A considerable fraction (at least 115.4-kbp, or 10.6%) of the 1.08-Mbp Wolbachia wBm genome has been transferred to its nematode host and retains high levels of similarity, including 227 wBm genes and gene fragments. Complete open reading frames were transferred for 32 of these genes, meaning they have the potential to produce functional proteins. Moreover, four transfers have evidence of life stage-specific regulation of transcription at levels similar to other nematode transcripts, strengthening the possibility that they are functional. CONCLUSIONS: There is extensive and ongoing transfer of Wolbachia DNA to the worm genome and some transfers are transcribed in a stage-specific manner at biologically relevant levels.

Transcriptional adaptation of pneumococci and human pharyngeal cells in the presence of a virus infection.

BACKGROUND: Viral upper respiratory tract infections are associated with increased colonization by Streptococcus pneumoniae but the mechanisms underlying this relationship are unclear. The objective of this study is to describe a comprehensive picture of the cellular interaction between the adhering bacteria and host cells in the presence or absence of a viral co-infection. RESULTS: Gene expression profiles of Detroit-562 pharyngeal cells, which were either mock-infected or infected with human respiratory syncytial virus (RSV) or human parainfluenza virus 3 (HPIV3), were analyzed using human microarrays. Transcription response of S. pneumoniae strain TIGR4 (serotype 4) in the presence of either mock- or viral-infected cells was analyzed by pneumococcal microarray. Significantly regulated genes were identified by both significance analysis of microarray (SAM) and a ≥ 2-fold change ratio cut-off. The adherence of S. pneumoniae to human pharyngeal cells was significantly augmented in the presence of RSV or HPIV3 infection. Global gene expression profiling of the host cells during infection with RSV or HPIV3 revealed increased transcription of carcinoembryonic antigen-related cell adhesion molecules (CEACAM1), CD47, fibronectin, interferon-stimulated genes and many other host cell adhesion molecules. Pneumococci increased transcription of several genes involved in adhesive functions (psaA, pilus islet), choline uptake and incorporation (lic operon), as well as transport and binding. CONCLUSIONS: We have identified a core transcriptome that represents the basic machinery required for adherence of pneumococci to D562 cells infected or not infected with a virus. These bacterial genes and cell adhesion molecules can potentially be used to control pneumococcal adherence occurring secondary to a viral infection.

Using Sybil for interactive comparative genomics of microbes on the web.

MOTIVATION: Analysis of multiple genomes requires sophisticated tools that provide search, visualization, interactivity and data export. Comparative genomics datasets tend to be large and complex, making development of these tools difficult. In addition to scalability, comparative genomics tools must also provide user-friendly interfaces such that the research scientist can explore complex data with minimal technical expertise. RESULTS: We describe a new version of the Sybil software package and its application to the important human pathogen Streptococcus pneumoniae. This new software provides a feature-rich set of comparative genomics tools for inspection of multiple genome structures, mining of orthologous gene families and identification of potential vaccine candidates. AVAILABILITY: The S.pneumoniae resource is online at http://strepneumo-sybil.igs.umaryland.edu. The software, database and website are available for download as a portable virtual machine and from http://sourceforge.net/projects/sybil.

Accumulation of endosymbiont genomes in an insect autosome followed by endosymbiont replacement.

Eukaryotic genomes can acquire bacterial DNA via lateral gene transfer (LGT).1 A prominent source of LGT is Wolbachia,2 a widespread endosymbiont of arthropods and nematodes that is transmitted maternally through female germline cells.3,4 The DNA transfer from the Wolbachia endosymbiont wAna to Drosophila ananassae is extensive5-7 and has been localized to chromosome 4, contributing to chromosome expansion in this lineage.6 As has happened frequently with claims of bacteria-to-eukaryote LGT, the contribution of wAna transfers to the expanded size of D. ananassae chromosome 4 has been specifically contested8 owing to an assembly where Wolbachia sequences were classified as contaminants and removed.9 Here, long-read sequencing with DNA from a Wolbachia-cured line enabled assembly of 4.9 Mbp of nuclear Wolbachia transfers (nuwts) in D. ananassae and a 24-kbp nuclear mitochondrial transfer. The nuwts are <8,000 years old in at least two locations in chromosome 4 with at least one whole-genome integration followed by rapid extensive duplication of most of the genome with regions that have up to 10 copies. The genes in nuwts are accumulating small indels and mobile element insertions. Among the highly duplicated genes are cifA and cifB, two genes associated with Wolbachia-mediated Drosophila cytoplasmic incompatibility. The wAna strain that was the source of nuwts was subsequently replaced by a different wAna endosymbiont. Direct RNA Nanopore sequencing of Wolbachia-cured lines identified nuwt transcripts, including spliced transcripts, but functionality, if any, remains elusive.

Whole-genome association study on tissue tropism phenotypes in group A Streptococcus.

Group A Streptococcus (GAS) has a rich evolutionary history of horizontal transfer among its core genes. Yet, despite extensive genetic mixing, GAS strains have discrete ecological phenotypes. To further our understanding of the molecular basis for ecological phenotypes, comparative genomic hybridization of a set of 97 diverse strains to a GAS pangenome microarray was undertaken, and the association of accessory genes with emm genotypes that define tissue tropisms for infection was determined. Of the 22 nonprophage accessory gene regions (AGRs) identified, only 3 account for all statistically significant linkage disequilibrium among strains having the genotypic biomarkers for throat versus skin infection specialists. Networked evolution and population structure analyses of loci representing each of the AGRs reveal that most strains with the skin specialist and generalist biomarkers form discrete clusters, whereas strains with the throat specialist biomarker are highly diverse. To identify coinherited and coselected accessory genes, the strength of genetic associations was determined for all possible pairwise combinations of accessory genes among the 97 GAS strains. Accessory genes showing very strong associations provide the basis for an evolutionary model, which reveals that a major transition between many throat and skin specialist haplotypes correlates with the gain or loss of genes encoding fibronectin-binding proteins. This study employs a novel synthesis of tools to help delineate the major genetic changes associated with key adaptive shifts in an extensively recombined bacterial species.

Improving pan-genome annotation using whole genome multiple alignment.

BACKGROUND: Rapid annotation and comparisons of genomes from multiple isolates (pan-genomes) is becoming commonplace due to advances in sequencing technology. Genome annotations can contain inconsistencies and errors that hinder comparative analysis even within a single species. Tools are needed to compare and improve annotation quality across sets of closely related genomes. RESULTS: We introduce a new tool, Mugsy-Annotator, that identifies orthologs and evaluates annotation quality in prokaryotic genomes using whole genome multiple alignment. Mugsy-Annotator identifies anomalies in annotated gene structures, including inconsistently located translation initiation sites and disrupted genes due to draft genome sequencing or pseudogenes. An evaluation of species pan-genomes using the tool indicates that such anomalies are common, especially at translation initiation sites. Mugsy-Annotator reports alternate annotations that improve consistency and are candidates for further review. CONCLUSIONS: Whole genome multiple alignment can be used to efficiently identify orthologs and annotation problem areas in a bacterial pan-genome. Comparisons of annotated gene structures within a species may show more variation than is actually present in the genome, indicating errors in genome annotation. Our new tool Mugsy-Annotator assists re-annotation efforts by highlighting edits that improve annotation consistency.