A molecular portrait of maternal sepsis from Byzantine Troy.

Pregnancy complications are poorly represented in the archeological record, despite their importance in contemporary and ancient societies. While excavating a Byzantine cemetery in Troy, we discovered calcified abscesses among a woman's remains. Scanning electron microscopy of the tissue revealed 'ghost cells', resulting from dystrophic calcification, which preserved ancient maternal, fetal and bacterial DNA of a severe infection, likely chorioamnionitis. Gardnerella vaginalis and Staphylococcus saprophyticus dominated the abscesses. Phylogenomic analyses of ancient, historical, and contemporary data showed that G. vaginalis Troy fell within contemporary genetic diversity, whereas S. saprophyticus Troy belongs to a lineage that does not appear to be commonly associated with human disease today. We speculate that the ecology of S. saprophyticus infection may have differed in the ancient world as a result of close contacts between humans and domesticated animals. These results highlight the complex and dynamic interactions with our microbial milieu that underlie severe maternal infections.

Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing.

Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, provide better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. Genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms.

Characterization of Genetic Variability of Venezuelan Equine Encephalitis Viruses.

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus that has caused large outbreaks of severe illness in both horses and humans. New approaches are needed to rapidly infer the origin of a newly discovered VEEV strain, estimate its equine amplification and resultant epidemic potential, and predict human virulence phenotype. We performed whole genome single nucleotide polymorphism (SNP) analysis of all available VEE antigenic complex genomes, verified that a SNP-based phylogeny accurately captured the features of a phylogenetic tree based on multiple sequence alignment, and developed a high resolution genome-wide SNP microarray. We used the microarray to analyze a broad panel of VEEV isolates, found excellent concordance between array- and sequence-based SNP calls, genotyped unsequenced isolates, and placed them on a phylogeny with sequenced genomes. The microarray successfully genotyped VEEV directly from tissue samples of an infected mouse, bypassing the need for viral isolation, culture and genomic sequencing. Finally, we identified genomic variants associated with serotypes and host species, revealing a complex relationship between genotype and phenotype.

Targeted amplification for enhanced detection of biothreat agents by next-generation sequencing.

BACKGROUND: Historically, identification of causal agents of disease has relied heavily on the ability to culture the organism in the laboratory and/or the use of pathogen-specific antibodies or sequence-based probes. However, these methods can be limiting: Even highly sensitive PCR-based assays must be continually updated due to signature degradation as new target strains and near neighbors are sequenced. Thus, there has been a need for assays that do not suffer as greatly from these limitations and/or biases. Recent advances in library preparation technologies for Next-Generation Sequencing (NGS) are focusing on the use of targeted amplification and targeted enrichment/capture to ensure that the most highly discriminating regions of the genomes of known targets (organism-unique regions and/or regions containing functionally important genes or phylogenetically-discriminating SNPs) will be sequenced, regardless of the complex sample background. RESULTS: In the present study, we have assessed the feasibility of targeted sequence enhancement via amplification to facilitate detection of a bacterial pathogen present in low copy numbers in a background of human genomic material. Our results indicate that the targeted amplification of signature regions can effectively identify pathogen genomic material present in as little as 10 copies per ml in a complex sample. Importantly, the correct species and strain calls could be made in amplified samples, while this was not possible in unamplified samples. CONCLUSIONS: The results presented here demonstrate the efficacy of a targeted amplification approach to biothreat detection, using multiple highly-discriminative amplicons per biothreat organism that provide redundancy in case of variation in some primer regions. Importantly, strain level discrimination was possible at levels of 10 genome equivalents. Similar results could be obtained through use of panels focused on the identification of amplicons targeted for specific genes or SNPs instead of, or in addition to, those targeted for specific organisms (ongoing gene-targeting work to be reported later). Note that without some form of targeted enhancement, the enormous background present in complex clinical and environmental samples makes it highly unlikely that sufficient coverage of key pathogen(s) present in the sample will be achieved with current NGS technology to guarantee that the most highly discriminating regions will be sequenced.

Using populations of human and microbial genomes for organism detection in metagenomes.

Identifying causative disease agents in human patients from shotgun metagenomic sequencing (SMS) presents a powerful tool to apply when other targeted diagnostics fail. Numerous technical challenges remain, however, before SMS can move beyond the role of research tool. Accurately separating the known and unknown organism content remains difficult, particularly when SMS is applied as a last resort. The true amount of human DNA that remains in a sample after screening against the human reference genome and filtering nonbiological components left from library preparation has previously been underreported. In this study, we create the most comprehensive collection of microbial and reference-free human genetic variation available in a database optimized for efficient metagenomic search by extracting sequences from GenBank and the 1000 Genomes Project. The results reveal new human sequences found in individual Human Microbiome Project (HMP) samples. Individual samples contain up to 95% human sequence, and 4% of the individual HMP samples contain 10% or more human reads. Left unidentified, human reads can complicate and slow down further analysis and lead to inaccurately labeled microbial taxa and ultimately lead to privacy concerns as more human genome data is collected.

Application of a pathogen microarray for the analysis of viruses and bacteria in clinical diagnostic samples from pigs.

Many of the disease syndromes challenging the commercial swine industry involve the analysis of complex problems caused by polymicrobial, emerging or reemerging, and transboundary pathogens. This study investigated the utility of the Lawrence Livermore Microbial Detection Array (Lawrence Livermore National Laboratory, Livermore, California), designed to detect 8,101 species of microbes, in the evaluation of known and unknown microbes in serum, oral fluid, and tonsil from pigs experimentally coinfected with Porcine reproductive and respiratory syndrome virus (PRRSV) and Porcine circovirus-2 (PCV-2). The array easily identified PRRSV and PCV-2, but at decreased sensitivities compared to standard polymerase chain reaction detection methods. The oral fluid sample was the most informative, possessing additional signatures for several swine-associated bacteria, including Streptococcus sp., Clostridium sp., and Staphylococcus sp.

kSNP3.0: SNP detection and phylogenetic analysis of genomes without genome alignment or reference genome.

UNLABELLED: We announce the release of kSNP3.0, a program for SNP identification and phylogenetic analysis without genome alignment or the requirement for reference genomes. kSNP3.0 is a significantly improved version of kSNP v2. AVAILABILITY AND IMPLEMENTATION: kSNP3.0 is implemented as a package of stand-alone executables for Linux and Mac OS X under the open-source BSD license. The executable packages, source code and a full User Guide are freely available at https://sourceforge.net/projects/ksnp/files/ CONTACT: barryghall@gmail.com.

Multiplex degenerate primer design for targeted whole genome amplification of many viral genomes.

Background. Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results. A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Each group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions. This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family.

Simulate_PCR for amplicon prediction and annotation from multiplex, degenerate primers and probes.

BACKGROUND: Pairing up primers to amplify desired targets and avoid undesired cross reactions can be a combinatorial challenge. Effective prediction of specificity and inclusivity from multiplexed primers and TaqMan®/Luminex® probes is a critical step in PCR design. RESULTS: Code is described to identify all primer and probe combinations from a list of unpaired, unordered candidates that should produce a product. It predicts and extracts all amplicon sequences in a large sequence database from a list of primers and probes, allowing degenerate bases and user-specified levels of primer-target mismatch tolerance. Amplicons hit by TaqMan®/Luminex® probes are indicated, and products may be annotated with gene information from NCBI. Fragment length distributions are calculated to predict electrophoretic gel banding patterns. CONCLUSIONS: Simulate_PCR is the only freely available software that can be run from the command line for high throughput applications which can calculate all products from large lists of primers and probes compared to a large sequence database such as nt. It requires no prior knowledge of how primers should be paired. Degenerate bases are allowed and entire amplicon sequences are extracted and annotated with gene information. Examples are provided for sets of TaqMan®/Luminex® PCR signatures predicted to amplify all HIV-1 genomes, all Coronaviridae genomes, and a group of antibiotic resistance genes. The software is a command line perl script freely available as open source.

The microbial detection array for detection of emerging viruses in clinical samples--a useful panmicrobial diagnostic tool.

Emerging viruses are usually endemic to tropical and sub-tropical regions of the world, but increased global travel, climate change and changes in lifestyle are believed to contribute to the spread of these viruses into new regions. Many of these viruses cause similar disease symptoms as other emerging viruses or common infections, making these unexpected pathogens difficult to diagnose. Broad-spectrum pathogen detection microarrays containing probes for all sequenced viruses and bacteria can provide rapid identification of viruses, guiding decisions about treatment and appropriate case management. We report a modified Whole Transcriptome Amplification (WTA) method that increases unbiased amplification, particular of RNA viruses. Using this modified WTA method, we tested the specificity and sensitivity of the Lawrence Livermore Microbial Detection Array (LLMDA) against a wide range of emerging viruses present in both non-clinical and clinical samples using two different microarray data analysis methods.

Microbial profiling of combat wound infection through detection microarray and next-generation sequencing.

Combat wound healing and resolution are highly affected by the resident microbial flora. We therefore sought to achieve comprehensive detection of microbial populations in wounds using novel genomic technologies and bioinformatics analyses. We employed a microarray capable of detecting all sequenced pathogens for interrogation of 124 wound samples from extremity injuries in combat-injured U.S. service members. A subset of samples was also processed via next-generation sequencing and metagenomic analysis. Array analysis detected microbial targets in 51% of all wound samples, with Acinetobacter baumannii being the most frequently detected species. Multiple Pseudomonas species were also detected in tissue biopsy specimens. Detection of the Acinetobacter plasmid pRAY correlated significantly with wound failure, while detection of enteric-associated bacteria was associated significantly with successful healing. Whole-genome sequencing revealed broad microbial biodiversity between samples. The total wound bioburden did not associate significantly with wound outcome, although temporal shifts were observed over the course of treatment. Given that standard microbiological methods do not detect the full range of microbes in each wound, these data emphasize the importance of supplementation with molecular techniques for thorough characterization of wound-associated microbes. Future application of genomic protocols for assessing microbial content could allow application of specialized care through early and rapid identification and management of critical patterns in wound bioburden.

Current strategic thinking for the development of a trivalent alphavirus vaccine for human use.

Vaccinations against the encephalitic alphaviruses (western, eastern, and Venezuelan equine encephalitis virus) are of significant interest to biological defense, public health, and agricultural communities alike. Although vaccines licensed for veterinary applications are used in the Western Hemisphere and attenuated or inactivated viruses have been used under Investigational New Drug status to protect at-risk personnel, there are currently no licensed vaccines for use in humans. Here, we will discuss the need for a trivalent vaccine that can protect humans against all three viruses, recent progress to such a vaccine, and a strategy to continue development to Food and Drug Administration licensure.

Screening of viral pathogens from pediatric ileal tissue samples after vaccination.

In 2010, researchers reported that the two US-licensed rotavirus vaccines contained DNA or DNA fragments from porcine circovirus (PCV). Although PCV, a common virus among pigs, is not thought to cause illness in humans, these findings raised several safety concerns. In this study, we sought to determine whether viruses, including PCV, could be detected in ileal tissue samples of children vaccinated with one of the two rotavirus vaccines. A broad spectrum, novel DNA detection technology, the Lawrence Livermore Microbial Detection Array (LLMDA), was utilized, and confirmation of viral pathogens using the polymerase chain reaction (PCR) was conducted. The LLMDA technology was recently used to identify PCV from one rotavirus vaccine. Ileal tissue samples were analyzed from 21 subjects, aged 15-62 months. PCV was not detected in any ileal tissue samples by the LLMDA or PCR. LLMDA identified a human rotavirus A from one of the vaccinated subjects, which is likely due to a recent infection from a wild type rotavirus. LLMDA also identified human parechovirus, a common gastroenteritis viral infection, from two subjects. Additionally, LLMDA detected common gastrointestinal bacterial organisms from the Enterobacteriaceae, Bacteroidaceae, and Streptococcaceae families from several subjects. This study provides a survey of viral and bacterial pathogens from pediatric ileal samples, and may shed light on future studies to identify pathogen associations with pediatric vaccinations.

When whole-genome alignments just won't work: kSNP v2 software for alignment-free SNP discovery and phylogenetics of hundreds of microbial genomes.

Effective use of rapid and inexpensive whole genome sequencing for microbes requires fast, memory efficient bioinformatics tools for sequence comparison. The kSNP v2 software finds single nucleotide polymorphisms (SNPs) in whole genome data. kSNP v2 has numerous improvements over kSNP v1 including SNP gene annotation; better scaling for draft genomes available as assembled contigs or raw, unassembled reads; a tool to identify the optimal value of k; distribution of packages of executables for Linux and Mac OS X for ease of installation and user-friendly use; and a detailed User Guide. SNP discovery is based on k-mer analysis, and requires no multiple sequence alignment or the selection of a single reference genome. Most target sets with hundreds of genomes complete in minutes to hours. SNP phylogenies are built by maximum likelihood, parsimony, and distance, based on all SNPs, only core SNPs, or SNPs present in some intermediate user-specified fraction of targets. The SNP-based trees that result are consistent with known taxonomy. kSNP v2 can handle many gigabases of sequence in a single run, and if one or more annotated genomes are included in the target set, SNPs are annotated with protein coding and other information (UTRs, etc.) from Genbank file(s). We demonstrate application of kSNP v2 on sets of viral and bacterial genomes, and discuss in detail analysis of a set of 68 finished E. coli and Shigella genomes and a set of the same genomes to which have been added 47 assemblies and four "raw read" genomes of H104:H4 strains from the recent European E. coli outbreak that resulted in both bloody diarrhea and hemolytic uremic syndrome (HUS), and caused at least 50 deaths.

Detection of Bacillus anthracis DNA in complex soil and air samples using next-generation sequencing.

Bacillus anthracis is the potentially lethal etiologic agent of anthrax disease, and is a significant concern in the realm of biodefense. One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracis genome, however, renders specific detection difficult, due to close homology with B. cereus and B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereus or B. thuringiensis. We also demonstrated the efficacy of a microbial census microarray in detecting B. anthracis in the same samples, representing a cost-effective and high-throughput approach, complementary to next-generation sequencing. Our results, in combination with the capacity of sequencing for providing insights into the genomic characteristics of complex and novel organisms, suggest that these platforms should be considered important components of a biosurveillance strategy.

Scalable metagenomic taxonomy classification using a reference genome database.

MOTIVATION: Deep metagenomic sequencing of biological samples has the potential to recover otherwise difficult-to-detect microorganisms and accurately characterize biological samples with limited prior knowledge of sample contents. Existing metagenomic taxonomic classification algorithms, however, do not scale well to analyze large metagenomic datasets, and balancing classification accuracy with computational efficiency presents a fundamental challenge. RESULTS: A method is presented to shift computational costs to an off-line computation by creating a taxonomy/genome index that supports scalable metagenomic classification. Scalable performance is demonstrated on real and simulated data to show accurate classification in the presence of novel organisms on samples that include viruses, prokaryotes, fungi and protists. Taxonomic classification of the previously published 150 giga-base Tyrolean Iceman dataset was found to take <20 h on a single node 40 core large memory machine and provide new insights on the metagenomic contents of the sample. AVAILABILITY: Software was implemented in C++ and is freely available at http://sourceforge.net/projects/lmat CONTACT: allen99@llnl.gov SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Optimizing SNP microarray probe design for high accuracy microbial genotyping.

Microarrays to characterize single nucleotide polymorphisms (SNPs) provide a cost-effective and rapid method (under 24h) to genotype microbes as an alternative to sequencing. We developed a pipeline for SNP discovery and microarray design that scales to 100's of microbial genomes. Here we tested various SNP probe design strategies against 8 sequenced isolates of Bacillus anthracis to compare sequence and microarray data. The best strategy allowed probe length to vary within 32-40 bp to equalize hybridization free energy. This strategy resulted in a call rate of 99.52% and concordance rate of 99.86% for finished genomes. Other probe design strategies averaged substantially lower call rates (94.65-96.41%) and slightly lower concordance rates (99.64-99.80%). These rates were lower for draft than finished genomes, consistent with higher incidence of sequencing errors and gaps. Highly accurate SNP calls were possible in complex soil and blood backgrounds down to 1000 copies, and moderately accurate SNP calls down to 100 spiked copies. The closest genome to the spiked strain was correctly identified at only 10 spiked copies. Discrepancies between sequence and array data did not alter the SNP-based phylogeny, regardless of the probe design strategy, indicating that SNP arrays can accurately place unsequenced isolates on a phylogeny.

Bioinformatics for microbial genotyping of equine encephalitis viruses, orthopoxviruses, and hantaviruses.

Microbial genotyping is essential for forensic discrimination of pathogen strains, tracing epidemics, and understanding evolutionary processes. Phylogenetic analyses were performed and genotyping assays designed for five viral species complexes or genera: Western, Eastern, and Venezuelan equine encephalitis viruses, hantavirus segments L, M, and S, and orthopoxviruses. For each group, sequence alignments and phylogenetic trees were built. PCR signatures composed of primer pairs or TaqMan™ triplets were designed and mapped to nodes of the trees for sub-type or strain specific PCR-based identification. In addition, single nucleotide polymorphisms (SNPs) were identified and mapped to trees, and SNP microarray probes were designed to enable highly multiplexed genotyping of an unsequenced sample by hybridization. SNP-based trees corresponded well with MSA trees. Near-perfect isolate resolution was possible for all viruses analyzed computationally using either SNPs or PCR signatures. More tree nodes were represented by SNP loci than by PCR signatures, as PCR signatures often represented subsets of strains not corresponding to a branch. However, while PCR genotyping is possible, the number of PCR signatures needed to characterize an unknown can be very large. SNP microarrays are a suitable alternative, as arrays enable highly multiplexed, high resolution genotyping of an unknown in a single hybridization assay.

Identification of SNPs associated with variola virus virulence.

BACKGROUND: Decades after the eradication of smallpox, its etiological agent, variola virus (VARV), remains a threat as a potential bioweapon. Outbreaks of smallpox around the time of the global eradication effort exhibited variable case fatality rates (CFRs), likely attributable in part to complex viral genetic determinants of smallpox virulence. We aimed to identify genome-wide single nucleotide polymorphisms associated with CFR. We evaluated unadjusted and outbreak geographic location-adjusted models of single SNPs and two- and three-way interactions between SNPs. FINDINGS: Using the data mining approach multifactor dimensionality reduction (MDR), we identified five VARV SNPs in models significantly associated with CFR. The top performing unadjusted model and adjusted models both revealed the same two-way gene-gene interaction. We discuss the biological plausibility of the influence of the SNPs identified these and other significant models on the strain-specific virulence of VARV. CONCLUSIONS: We have identified genetic loci in the VARV genome that are statistically associated with VARV virulence as measured by CFR. While our ability to infer a causal relationship between the specific SNPs identified in our analysis and VARV virulence is limited, our results suggest that smallpox severity is in part associated with VARV strain variation and that VARV virulence may be determined by multiple genetic loci. This study represents the first application of MDR to the identification of pathogen gene-gene interactions for predicting infectious disease outbreak severity.

Skip the alignment: degenerate, multiplex primer and probe design using K-mer matching instead of alignments.

PriMux is a new software package for selecting multiplex compatible, degenerate primers and probes to detect diverse targets such as viruses. It requires no multiple sequence alignment, instead applying k-mer algorithms, hence it scales well for large target sets and saves user effort from curating sequences into alignable groups. PriMux has the capability to predict degenerate primers as well as probes suitable for TaqMan or other primer/probe triplet assay formats, or simply probes for microarray or other single-oligo assay formats. PriMux employs suffix array methods for efficient calculations on oligos 10-~100 nt in length. TaqMan® primers and probes for each segment of Rift Valley fever virus were designed using PriMux, and lab testing comparing signatures designed using PriMux versus those designed using traditional methods demonstrated equivalent or better sensitivity for the PriMux-designed signatures compared to traditional signatures. In addition, we used PriMux to design TaqMan® primers and probes for unalignable or poorly alignable groups of targets: that is, all segments of Rift Valley fever virus analyzed as a single target set of 198 sequences, or all 2863 Dengue virus genomes for all four serotypes available at the time of our analysis. The PriMux software is available as open source from http://sourceforge.net/projects/PriMux.