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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

LAVA: an open-source approach to designing LAMP (loop-mediated isothermal amplification) DNA signatures.

Background: We developed an extendable open-source Loop-mediated isothermal AMPlification (LAMP) signature design program called LAVA (LAMP Assay Versatile Analysis). LAVA was created in response to limitations of existing LAMP signature programs.

Results: LAVA identifies combinations of six primer regions for basic LAMP signatures, or combinations of eight primer regions for LAMP signatures with loop primers, which can be used as LAMP signatures.

A microbial detection array (MDA) for viral and bacterial detection.

Background: Identifying the bacteria and viruses present in a complex sample is useful in disease diagnostics, product safety, environmental characterization, and research. Array-based methods have proven utility to detect in a single assay at a reasonable cost any microbe from the thousands that have been sequenced.

Methods: We designed a pan-Microbial Detection Array (MDA) to detect all known viruses (including phages), bacteria and plasmids and developed a novel statistical analysis method to identify mixtures of organisms from complex samples hybridized to the array.

Multiplex primer prediction software for divergent targets.

We describe a Multiplex Primer Prediction (MPP) algorithm to build multiplex compatible primer sets to amplify all members of large, diverse and unalignable sets of target sequences. The MPP algorithm is scalable to larger target sets than other available software, and it does not require a multiple sequence alignment. We applied it to questions in viral detection, and demonstrated that there are no universally conserved priming sequences among viruses and that it could require an unfeasibly large number of primers ( approximately 3700 18-mers or approximately 2000 10-mers) to generate amplicons from all sequenced viruses.

Conserved amino acid markers from past influenza pandemic strains.

Background: Finding the amino acid mutations that affect the severity of influenza infections remains an open and challenging problem. Of special interest is better understanding how current circulating influenza strains could evolve into a new pandemic strain. Influenza proteomes from distinct viral phenotype classes were searched for class specific amino acid mutations conserved in past pandemics, using reverse engineered linear classifiers.

Predicting the sensitivity and specificity of published real-time PCR assays.

Background: In recent years real-time PCR has become a leading technique for nucleic acid detection and quantification. These assays have the potential to greatly enhance efficiency in the clinical laboratory. Choice of primer and probe sequences is critical for accurate diagnosis in the clinic, yet current primer/probe signature design strategies are limited, and signature evaluation methods are lacking.