PAIPline: pathogen identification in metagenomic and clinical next generation sequencing samples.

Motivation: Next generation sequencing (NGS) has provided researchers with a powerful tool to characterize metagenomic and clinical samples in research and diagnostic settings. NGS allows an open view into samples useful for pathogen detection in an unbiased fashion and without prior hypothesis about possible causative agents. However, NGS datasets for pathogen detection come with different obstacles, such as a very unfavorable ratio of pathogen to host reads. Alongside often appearing false positives and irrelevant organisms, such as contaminants, tools are often challenged by samples with low pathogen loads and might not report organisms present below a certain threshold. Furthermore, some metagenomic profiling tools are only focused on one particular set of pathogens, for example bacteria. Results: We present PAIPline, a bioinformatics pipeline specifically designed to address problems associated with detecting pathogens in diagnostic samples. PAIPline particularly focuses on userfriendliness and encapsulates all necessary steps from preprocessing to resolution of ambiguous reads and filtering up to visualization in a single tool. In contrast to existing tools, PAIPline is more specific while maintaining sensitivity. This is shown in a comparative evaluation where PAIPline was benchmarked along other well-known metagenomic profiling tools on previously published well-characterized datasets. Additionally, as part of an international cooperation project, PAIPline was applied to an outbreak sample of hemorrhagic fevers of then unknown etiology. The presented results show that PAIPline can serve as a robust, reliable, user-friendly, adaptable and generalizable stand-alone software for diagnostics from NGS samples and as a stepping stone for further downstream analyses. Availability and implementation: PAIPline is freely available under https://gitlab.com/rki_bioinformatics/paipline.

PriLive: privacy-preserving real-time filtering for next-generation sequencing.

Motivation: In next-generation sequencing, re-identification of individuals and other privacy-breaching strategies can be applied even for anonymized data. This also holds true for applications in which human DNA is acquired as a by-product, e.g. for viral or metagenomic samples from a human host. Conventional data protection strategies including cryptography and post-hoc filtering are only appropriate for the final and processed sequencing data. This can result in an insufficient level of data protection and a considerable time delay in the further analysis workflow. Results: We present PriLive, a novel tool for the automated removal of sensitive data while the sequencing machine is running. Thereby, human sequence information can be detected and removed before being completely produced. This facilitates the compliance with strict data protection regulations. The unique characteristic to cause almost no time delay for further analyses is also a clear benefit for applications other than data protection. Especially if the sequencing data are dominated by known background signals, PriLive considerably accelerates consequent analyses by having only fractions of input data. Besides these conceptual advantages, PriLive achieves filtering results at least as accurate as conventional post-hoc filtering tools. Availability and implementation: PriLive is open-source software available at https://gitlab.com/rki_bioinformatics/PriLive. Supplementary information: Supplementary data are available at Bioinformatics online.

seq-seq-pan: building a computational pan-genome data structure on whole genome alignment.

BACKGROUND: The increasing application of next generation sequencing technologies has led to the availability of thousands of reference genomes, often providing multiple genomes for the same or closely related species. The current approach to represent a species or a population with a single reference sequence and a set of variations cannot represent their full diversity and introduces bias towards the chosen reference. There is a need for the representation of multiple sequences in a composite way that is compatible with existing data sources for annotation and suitable for established sequence analysis methods. At the same time, this representation needs to be easily accessible and extendable to account for the constant change of available genomes. RESULTS: We introduce seq-seq-pan, a framework that provides methods for adding or removing new genomes from a set of aligned genomes and uses these to construct a whole genome alignment. Throughout the sequential workflow the alignment is optimized for generating a representative linear presentation of the aligned set of genomes, that enables its usage for annotation and in downstream analyses. CONCLUSIONS: By providing dynamic updates and optimized processing, our approach enables the usage of whole genome alignment in the field of pan-genomics. In addition, the sequential workflow can be used as a fast alternative to existing whole genome aligners for aligning closely related genomes. seq-seq-pan is freely available at https://gitlab.com/rki_bioinformatics.

HiLive: real-time mapping of illumina reads while sequencing.

Motivation: Next Generation Sequencing is increasingly used in time critical, clinical applications. While read mapping algorithms have always been optimized for speed, they follow a sequential paradigm and only start after finishing of the sequencing run and conversion of files. Since Illumina machines write intermediate output results, HiLive performs read mapping while still sequencing and thereby drastically reduces crucial overall sample analysis time, e.g. in precision medicine. Methods: We present HiLive as a novel real time read mapper that implements a k-mer based alignment strategy. HiLive continuously reads intermediate BCL files produced by Illumina sequencers and then extends initial k-mer matches by increasingly produced data from the sequencer. Results: We applied HiLive on real human transcriptome data to show that final read alignments are reported within few minutes after the end of a full Illumina HiSeq 1500 run, while already the necessary conversion to FASTQ files as the standard input to current read mapping methods takes roughly five times as long. Further, we show on simulated and real data that HiLive has comparable accuracy to recent read mappers. Availability and Implementation: HiLive and its source code are freely available from https://gitlab.com/SimonHTausch/HiLive . Contact: renardB@rki.de. Supplementary information: Supplementary data are available at Bioinformatics online.

Berlin Squirrelpox Virus, a New Poxvirus in Red Squirrels, Berlin, Germany.

Near Berlin, Germany, several juvenile red squirrels (Sciurus vulgaris) were found with moist, crusty skin lesions. Histology, electron microscopy, and cell culture isolation revealed an orthopoxvirus-like infection. Subsequent PCR and genome analysis identified a new poxvirus (Berlin squirrelpox virus) that could not be assigned to any known poxvirus genera.

Protocol for metagenomic virus detection in clinical specimens.

Sixty percent of emerging viruses have a zoonotic origin, making transmission from animals a major threat to public health. Prompt identification and analysis of these pathogens are indispensable to taking action toward prevention and protection of the affected population. We quantifiably compared classical and modern approaches of virus purification and enrichment in theory and experiments. Eventually, we established an unbiased protocol for detection of known and novel emerging viruses from organ tissues (tissue-based universal virus detection for viral metagenomics [TUViD-VM]). The final TUViD-VM protocol was extensively validated by using real-time PCR and next-generation sequencing. We could increase the amount of detectable virus nucleic acids and improved the detection of viruses <75,000-fold compared with other tested approaches. This TUViD-VM protocol can be used in metagenomic and virome studies to increase the likelihood of detecting viruses from any biological source.

Identification and characterization of episomal forms of integrative genomic islands in the genus Francisella.

Recently, we identified a putative prophage on a genomic island (GI) within the genome sequence of Francisella hispaniensis isolate AS0-814 (Francisella tularensis subsp. novicida-like 3523) by the analysis of the CRISPR-Cas systems of Francisella. Various spacer DNAs within the CRISPR region of different F. tularensis subsp. novicida strains were found to be homologous to the putative prophage (Schunder et al., 2013, Int. J. Med. Microbiol. 303:51-60). Now we identified the GI (FhaGI-1) as a mobile element which is able to form a circular episomal structure. The circular episomal form of FhaGI-1 is generated by F. hispaniensis, and the excision of the island is an integrase-dependent and site-specific process. Furthermore, we could demonstrate that the excision of the island is also possible in other bacterial species (Escherichia coli). In addition, we could show that a genetically generated small variant of the island is also functional and, after its electroporation into strain F. tularensis subsp. holarctica LVS, the GI was stable and site-specifically integrated into the genome of the transformants. The integrase is sufficient for the integration and excision of the small variant into and from the DNA backbone, respectively. Thus, the element may be suitable to be used as a genetic tool in F. tularensis research. Furthermore, we identified the tRNA(Val) gene of Francisella as an integration site for GIs. Genomic island FphGI-1 was identified in Francisella philomiragia ATCC 25016. We were not able to detect the episomal form of this GI, probably due to a mutated attR site. However, we could demonstrate that integrative GIs are present in Francisella and that they may allow horizontal gene transfer between different Francisella species.

Complete genome sequence of the porcine isolate Enterococcus faecalis D32.

The complete and annotated genome sequence of Enterococcus faecalis D32, a commensal strain isolated from a Danish pig, suggests putative adaptation to the porcine host and absence of distinct virulence-associated traits.

Rapid detection of anti-Vaccinia virus neutralizing antibodies.

Increasing infections with Monkeypox and Cowpox viruses pose a continuous and growing threat to human health. The standard method for detecting poxvirus neutralizing antibodies is the plaque-reduction neutralization test that is specific but also time-consuming and laborious. Therefore, a rapid and reliable method was developed to determine neutralizing antibody titers within twelve hours. The new assay measures viral mRNA transcription as a marker for actively replicating virus after incomplete neutralization using real-time PCR.

Infection of human airway epithelial cells by different subtypes of Dobrava-Belgrade virus reveals gene expression patterns corresponding to their virulence potential.

Dobrava-Belgrade virus (DOBV) is a pathogen causing hemorrhagic fever with renal syndrome in Europe. Virulence and case fatality rate are associated with virus genotype; however the reasons for these differences are not well understood. In this work we present virus-specific effects on the gene expression profiles of human lung epithelial cells (A549) infected with different genotypes of DOBV (Dobrava, Kurkino, and Sochi), as well as the low-virulent Tula virus (TULV). The data was collected by whole-genome gene expression microarrays and confirmed by quantitative real-time PCR. Despite their close genetic relationship, the expression profiles induced by infection with different hantaviruses are significantly varying. Major differences were observed in regulation of immune response genes, which were especially induced by highly virulent DOBV genotypes Dobrava and Sochi in contrast to less virulent DOBV-Kurkino and TULV. This work gives first insights into the differences of virus - host interactions of DOBV on genotype level.

Investigating the zoonotic origin of the West African Ebola epidemic.

The severe Ebola virus disease epidemic occurring in West Africa stems from a single zoonotic transmission event to a 2-year-old boy in Meliandou, Guinea. We investigated the zoonotic origins of the epidemic using wildlife surveys, interviews, and molecular analyses of bat and environmental samples. We found no evidence for a concurrent outbreak in larger wildlife. Exposure to fruit bats is common in the region, but the index case may have been infected by playing in a hollow tree housing a colony of insectivorous free-tailed bats (Mops condylurus). Bats in this family have previously been discussed as potential sources for Ebola virus outbreaks, and experimental data have shown that this species can survive experimental infection. These analyses expand the range of possible Ebola virus sources to include insectivorous bats and reiterate the importance of broader sampling efforts for understanding Ebola virus ecology.