Sensitivity and breadth of detection of high-throughput sequencing for adventitious virus detection.

High-throughput sequencing (HTS) is capable of broad virus detection encompassing both known and unknown adventitious viruses in a variety of sample matrices. We describe the development of a general-purpose HTS-based method for the detection of adventitious viruses. Performance was evaluated using 16 viruses equivalent to well-characterized National Institutes of Health (NIH) virus stocks and another six viruses of interest.

Considerations for Optimization of High-Throughput Sequencing Bioinformatics Pipelines for Virus Detection.

High-throughput sequencing (HTS) has demonstrated capabilities for broad virus detection based upon discovery of known and novel viruses in a variety of samples, including clinical, environmental, and biological. An important goal for HTS applications in biologics is to establish parameter settings that can afford adequate sensitivity at an acceptable computational cost (computation time, computer memory, storage, expense or/and efficiency), at critical steps in the bioinformatics pipeline, including initial data quality assessment, trimming/cleaning, and assembly (to reduce data volume and increase likelihood of appropriate sequence identification). Additionally, the quality and reliability of the results depend on the availability of a complete and curated viral database for obtaining accurate results; selection of sequence alignment programs and their configuration, that retains specificity for broad virus detection with reduced false-positive signals; removal of host sequences without loss of endogenous viral sequences of interest; and use of a meaningful reporting format, which can retain critical information of the analysis for presentation of readily interpretable data and actionable results.

A Multicenter Study To Evaluate the Performance of High-Throughput Sequencing for Virus Detection.

The capability of high-throughput sequencing (HTS) for detection of known and unknown viruses makes it a powerful tool for broad microbial investigations, such as evaluation of novel cell substrates that may be used for the development of new biological products. However, like any new assay, regulatory applications of HTS need method standardization. Therefore, our three laboratories initiated a study to evaluate performance of HTS for potential detection of viral adventitious agents by spiking model viruses in different cellular matrices to mimic putative materials for manufacturing of biologics.

Advanced Virus Detection Technologies Interest Group (AVDTIG): Efforts on High Throughput Sequencing (HTS) for Virus Detection.

Several nucleic-acid based technologies have recently emerged with capabilities for broad virus detection. One of these, high throughput sequencing, has the potential for novel virus detection because this method does not depend upon prior viral sequence knowledge. However, the use of high throughput sequencing for testing biologicals poses greater challenges as compared to other newly introduced tests due to its technical complexities and big data bioinformatics.

Cataloguing the taxonomic origins of sequences from a heterogeneous sample using phylogenomics: applications in adventitious agent detection.

Unlabelled: We have designed and implemented a software system, named PhyloID™, that can be used to detect putative adventitious agents in biological samples characterized by next-generation sequencing. PhyloID is run in two steps, each being a self-contained automated process amenable to GMP validation. The first module, MiLY, is responsible for assembling individual sequence reads into contigs, and annotating all sequences with a unique sequence identifier, the number of reads in each contig, and the length of the sequence.

Preliminary Evaluation of Next-Generation Sequencing Performance Relative to qPCR and In Vitro Cell Culture Tests for Human Cytomegalovirus.

Unlabelled: To compare the performances of conventional in vitro indicator cell culture, quantitative polymerase chain reaction (qPCR), and next-generation sequencing (NGS) as detection methods for adventitious agents, a preliminary assessment was performed using human cytomegalovirus (HCMV) as a model virus. HCMV was spiked into a crude viral harvest at various concentrations and inoculated onto MRC-5 cell monolayers. The cultures were observed for cytopathic effects (CPEs) as per the compendial method requirements, and samples were taken at various time points for analysis by qPCR or NGS.

Coevolution reveals a network of human proteins originating with multicellularity.

Protein interaction networks play central roles in biological systems, from simple metabolic pathways through complex programs permitting the development of organisms. Multicellularity could only have arisen from a careful orchestration of cellular and molecular roles and responsibilities, all properly controlled and regulated. Disease reflects a breakdown of this organismal homeostasis.

Using coevolution to predict protein-protein interactions.

Bioinformatic methods to predict protein-protein interactions (PPI) via coevolutionary analysis have -positioned themselves to compete alongside established in vitro methods, despite a lack of understanding for the underlying molecular mechanisms of the coevolutionary process. Investigating the alignment of coevolutionary predictions of PPI with experimental data can focus the effective scope of prediction and lead to better accuracies. A new rate-based coevolutionary method, MMM, preferentially finds obligate interacting proteins that form complexes, conforming to results from studies based on coimmunoprecipitation coupled with mass spectrometry.

The human protein coevolution network.

Coevolution maintains interactions between phenotypic traits through the process of reciprocal natural selection. Detecting molecular coevolution can expose functional interactions between molecules in the cell, generating insights into biological processes, pathways, and the networks of interactions important for cellular function. Prediction of interaction partners from different protein families exploits the property that interacting proteins can follow similar patterns and relative rates of evolution.

The impact of reticulate evolution on genome phylogeny.

Genome phylogenies are used to build tree-like representations of evolutionary relationships among genomes. However, in condensing the phylogenetic signals within a set of genomes down to a single tree, these methods generally do not explicitly take into account discordant signals arising due to lateral genetic transfer. Because conflicting vertical and horizontal signals can produce compromise trees that do not reflect either type of history, it is essential to understand the sensitivity of inferred genome phylogenies to these confounding effects.

Integron-associated gene cassettes in Halifax Harbour: assessment of a mobile gene pool in marine sediments.

The integron/gene cassette systems identified in bacteria comprise a class of genetic elements that allow adaptation by acquisition of gene cassettes. Integron gene cassettes have been shown to facilitate the spread of drug resistance in human pathogens but their role outside a clinical setting has not been explored extensively. We sequenced 2145 integron gene cassettes from four marine sediment samples taken from the vicinity of Halifax Nova Scotia, Canada, increasing the number of gene cassettes obtained from environmental microbial communities by 10-fold.

A simulation test bed for hypotheses of genome evolution.

Motivation: Microbial genomes undergo evolutionary processes such as gene family expansion and contraction, variable rates and patterns of sequence substitution and lateral genetic transfer. Simulation tools are essential for both the generation of data under different evolutionary models and the validation of analytical methods on such data. However, meaningful investigation of phenomena such as lateral genetic transfer requires the simultaneous consideration of many underlying evolutionary processes.

Phylogenetic analyses of cyanobacterial genomes: quantification of horizontal gene transfer events.

Using 1128 protein-coding gene families from 11 completely sequenced cyanobacterial genomes, we attempt to quantify horizontal gene transfer events within cyanobacteria, as well as between cyanobacteria and other phyla. A novel method of detecting and enumerating potential horizontal gene transfer events within a group of organisms based on analyses of "embedded quartets" allows us to identify phylogenetic signal consistent with a plurality of gene families, as well as to delineate cases of conflict to the plurality signal, which include horizontally transferred genes. To infer horizontal gene transfer events between cyanobacteria and other phyla, we added homologs from 168 available genomes.

Ancient lateral gene transfer in the evolution of Bdellovibrio bacteriovorus.

The recently sequenced genome of the predatory delta-proteobacterium Bdellovibrio bacteriovorus provides many insights into its metabolism and evolution. Because its genes are reasonably uniform in G+C content, it was suggested that B. bacteriovorus actively resists recombination with foreign DNA and horizontal transfer of DNA from other bacteria.

The two tempos of nuclear pore complex evolution: highly adapting proteins in an ancient frozen structure.

Background: The origin of the nuclear compartment has been extensively debated, leading to several alternative views on the evolution of the eukaryotic nucleus. Until recently, too little phylogenetic information was available to address this issue by using multiple characters for many lineages.

Results: We analyzed 65 proteins integral to or associated with the nuclear pore complex (NPC), including all the identified nucleoporins, the components of their anchoring system and some of their main partners.

Deduction of probable events of lateral gene transfer through comparison of phylogenetic trees by recursive consolidation and rearrangement.

Background: When organismal phylogenies based on sequences of single marker genes are poorly resolved, a logical approach is to add more markers, on the assumption that weak but congruent phylogenetic signal will be reinforced in such multigene trees. Such approaches are valid only when the several markers indeed have identical phylogenies, an issue which many multigene methods (such as the use of concatenated gene sequences or the assembly of supertrees) do not directly address. Indeed, even when the true history is a mixture of vertical descent for some genes and lateral gene transfer (LGT) for others, such methods produce unique topologies.

GANN: genetic algorithm neural networks for the detection of conserved combinations of features in DNA.

Background: The multitude of motif detection algorithms developed to date have largely focused on the detection of patterns in primary sequence. Since sequence-dependent DNA structure and flexibility may also play a role in protein-DNA interactions, the simultaneous exploration of sequence- and structure-based hypotheses about the composition of binding sites and the ordering of features in a regulatory region should be considered as well. The consideration of structural features requires the development of new detection tools that can deal with data types other than primary sequence.

Weighted genome trees: refinements and applications.

There are many ways to group completed genome sequences in hierarchical patterns (trees) reflecting relationships between their genes. Such groupings help us organize biological information and bear crucially on underlying processes of genome and organismal evolution. Genome trees make use of all comparable genes but can variously weight the contributions of these genes according to similarity, congruent patterns of similarity, or prevalence among genomes.

Computing prokaryotic gene ubiquity: rescuing the core from extinction.

The genomic core concept has found several uses in comparative and evolutionary genomics. Defined as the set of all genes common to (ubiquitous among) all genomes in a phylogenetically coherent group, core size decreases as the number and phylogenetic diversity of the relevant group increases. Here, we focus on methods for defining the size and composition of the core of all genes shared by sequenced genomes of prokaryotes (Bacteria and Archaea).

Characterization of species-specific genes using a flexible, web-based querying system.

We describe a query-based web-accessible system (www.neurogadgets.com/bws.

Distributional profiles of homologous open reading frames among bacterial phyla: implications for vertical and lateral transmission.

If open reading frames (ORFs) have been transmitted primarily by vertical descent, the distributional profile of orthologues of each ORF should be congruent with the organismal tree or a subtree thereof. Distributional patterns not reconciled parsimoniously with tree-like descent and loss are prima facie evidence of lateral gene transfer. Herein, a rigorous criterion for recognizing ORF distributions is described and implemented; it does not require the inference of phylogenetic trees, nor does it assume any specific tree.

Inferring genome trees by using a filter to eliminate phylogenetically discordant sequences and a distance matrix based on mean normalized BLASTP scores.

Darwin's paradigm holds that the diversity of present-day organisms has arisen via a process of genetic descent with modification, as on a bifurcating tree. Evidence is accumulating that genes are sometimes transferred not along lineages but rather across lineages. To the extent that this is so, Darwin's paradigm can apply only imperfectly to genomes, potentially complicating or perhaps undermining attempts to reconstruct historical relationships among genomes (i.