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September 22, 2019  |  

Acidipropionibacterium virtanenii sp. nov., isolated from malted barley.

A Gram-stain-positive, catalase-positive and pleomorphic rod organism was isolated from malted barley in Finland, classified initially by partial 16S rRNA gene sequencing and originally deposited in the VTT Culture Collection as a strain of Propionibacterium acidipropionici (currently Acidipropionibacterium acidipropionici). The subsequent comparison of the whole 16S rRNA gene with other representatives of the genus Acidipropionibacterium revealed that the strain belongs to a novel species, most closely related to Acidipropionibacterium microaerophilum and Acidipropionibacterium acidipropionici, with similarity values of 98.46 and 98.31?%, respectively. The whole genome sequencing using PacBio RS II platform allowed further comparison of the genome with all of the other DNA sequences available for the type strains of the Acidipropionibacterium species. Those comparisons revealed the highest similarity of strain JS278T to A. acidipropionici, which was confirmed by the average nucleotide identity analysis. The genome of strain JS278T is intermediate in size compared to the A. acidipropionici and Acidipropionibacterium jensenii at 3?432?872?bp, the G+C?content is 68.4?mol%. The strain fermented a wide range of carbon sources, and produced propionic acid as the major fermentation product. Besides its poor ability to grow at 37?°C and positive catalase reaction, the observed phenotype was almost indistinguishable from those of A. acidipropionici and A. jensenii. Based on our findings, we conclude that the organism represents a novel member of the genus Acidipropionibacterium, for which we propose the name Acidipropionibacteriumvirtanenii sp. nov. The type strain is JS278T (=VTT E-113202T=DSM 106790T).

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September 22, 2019  |  

Transcriptional adaptations during long-term persistence of Staphylococcus aureus in the airways of a cystic fibrosis patient.

The lungs of Cystic fibrosis (CF) patients are often colonized and/or infected by Staphylococcus aureus for years, mostly by one predominant clone. For long-term survival in this environment, S. aureus needs to adapt during its interactions with host factors, antibiotics, and other pathogens. Here, we study long-term transcriptional as well as genomic adaptations of an isogenic pair of S. aureus isolates from a single patient using RNA sequencing (RNA-Seq) and whole genome sequencing (WGS). Mimicking in vivo conditions, we cultivated the S. aureus isolates using artificial sputum medium before harvesting RNA for subsequent analysis. We confirmed our RNA-Seq data using quantitative real-time (qRT)-PCR and additionally investigated intermediate isolates from the same patient representing in total 13.2 years of persistence in the CF airways. Comparative RNA-Seq analysis of the first and the last (“late”) isolate revealed significant differences in the late isolate after 13.2 years of persistence. Of the 2545 genes expressed in both isolates that were cultivated aerobically, 256 genes were up- and 161 were down-regulated with a minimum 2-fold change (2f). Focusing on 25 highly (=8f) up- (n=9) or down- (n=16) regulated genes, we identified several genes encoding for virulence factors involved in immune evasion, bacterial spread or secretion (e.g. spa, sak, and esxA). Moreover, these genes displayed similar expression trends under aerobic, microaerophilic and anaerobic conditions. Further qRT-PCR-experiments of highly up- or down-regulated genes within intermediate S. aureus isolates resulted in different gene expression patterns over the years. Using sequencing analysis of the differently expressed genes and their upstream regions in the late S. aureus isolate resulted in only few genomic alterations. Comparative transcriptomic analysis revealed adaptive changes affecting mainly genes involved in host-pathogen interaction. Although the underlying mechanisms were not known, our results suggest adaptive processes beyond genomic mutations triggered by local factors rather than by activation of global regulators. Copyright © 2014 The Authors. Published by Elsevier GmbH.. All rights reserved.

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September 22, 2019  |  

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area.

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.

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September 22, 2019  |  

Clinical PathoScope: rapid alignment and filtration for accurate pathogen identification in clinical samples using unassembled sequencing data.

The use of sequencing technologies to investigate the microbiome of a sample can positively impact patient healthcare by providing therapeutic targets for personalized disease treatment. However, these samples contain genomic sequences from various sources that complicate the identification of pathogens.Here we present Clinical PathoScope, a pipeline to rapidly and accurately remove host contamination, isolate microbial reads, and identify potential disease-causing pathogens. We have accomplished three essential tasks in the development of Clinical PathoScope. First, we developed an optimized framework for pathogen identification using a computational subtraction methodology in concordance with read trimming and ambiguous read reassignment. Second, we have demonstrated the ability of our approach to identify multiple pathogens in a single clinical sample, accurately identify pathogens at the subspecies level, and determine the nearest phylogenetic neighbor of novel or highly mutated pathogens using real clinical sequencing data. Finally, we have shown that Clinical PathoScope outperforms previously published pathogen identification methods with regard to computational speed, sensitivity, and specificity.Clinical PathoScope is the only pathogen identification method currently available that can identify multiple pathogens from mixed samples and distinguish between very closely related species and strains in samples with very few reads per pathogen. Furthermore, Clinical PathoScope does not rely on genome assembly and thus can more rapidly complete the analysis of a clinical sample when compared with current assembly-based methods. Clinical PathoScope is freely available at: http://sourceforge.net/projects/pathoscope/.

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September 22, 2019  |  

Evolution of selective-sequencing approaches for virus discovery and virome analysis.

Recent advances in sequencing technologies have transformed the field of virus discovery and virome analysis. Once mostly confined to the traditional Sanger sequencing based individual virus discovery, is now entirely replaced by high throughput sequencing (HTS) based virus metagenomics that can be used to characterize the nature and composition of entire viromes. To better harness the potential of HTS for the study of viromes, sample preparation methodologies use different approaches to exclude amplification of non-viral components that can overshadow low-titer viruses. These virus-sequence enrichment approaches mostly focus on the sample preparation methods, like enzymatic digestion of non-viral nucleic acids and size exclusion of non-viral constituents by column filtration, ultrafiltration or density gradient centrifugation. However, recently a new approach of virus-sequence enrichment called virome-capture sequencing, focused on the amplification or HTS library preparation stage, was developed to increase the ability of virome characterization. This new approach has the potential to further transform the field of virus discovery and virome analysis, but its technical complexity and sequence-dependence warrants further improvements. In this review we discuss the different methods, their applications and evolution, for selective sequencing based virome analysis and also propose refinements needed to harness the full potential of HTS for virome analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

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September 22, 2019  |  

Survey of Ixodes pacificus ticks in California reveals a diversity of microorganisms and a novel and widespread Anaplasmataceae species.

Ixodes pacificus ticks can harbor a wide range of human and animal pathogens. To survey the prevalence of tick-borne known and putative pathogens, we tested 982 individual adult and nymphal I. pacificus ticks collected throughout California between 2007 and 2009 using a broad-range PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) assay designed to detect a wide range of tick-borne microorganisms. Overall, 1.4% of the ticks were found to be infected with Borrelia burgdorferi, 2.0% were infected with Borrelia miyamotoi and 0.3% were infected with Anaplasma phagocytophilum. In addition, 3.0% were infected with Babesia odocoilei. About 1.2% of the ticks were co-infected with more than one pathogen or putative pathogen. In addition, we identified a novel Anaplasmataceae species that we characterized by sequencing of its 16S rRNA, groEL, gltA, and rpoB genes. Sequence analysis indicated that this organism is phylogenetically distinct from known Anaplasma species with its closest genetic near neighbors coming from Asia. The prevalence of this novel Anaplasmataceae species was as high as 21% at one site, and it was detected in 4.9% of ticks tested statewide. Based upon this genetic characterization we propose that this organism be called ‘Candidatus Cryptoplasma californiense’. Knowledge of this novel microbe will provide awareness for the community about the breadth of the I. pacificus microbiome, the concept that this bacterium could be more widely spread; and an opportunity to explore whether this bacterium also contributes to human or animal disease burden.

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September 22, 2019  |  

Discovery of a divergent HPIV4 from respiratory secretions using second and third generation metagenomic sequencing.

Molecular detection of viruses has been aided by high-throughput sequencing, permitting the genomic characterization of emerging strains. In this study, we comprehensively screened 500 respiratory secretions from children with upper and/or lower respiratory tract infections for viral pathogens. The viruses detected are described, including a divergent human parainfluenza virus type 4 from GS FLX pyrosequencing of 92 specimens. Complete full-genome characterization of the virus followed, using Single Molecule, Real-Time (SMRT) sequencing. Subsequent “primer walking” combined with Sanger sequencing validated the RS platform’s utility in viral sequencing from complex clinical samples. Comparative genomics reveals the divergent strain clusters with the only completely sequenced HPIV4a subtype. However, it also exhibits various structural features present in one of the HPIV4b reference strains, opening questions regarding their lifecycle and evolutionary relationships among these viruses. Clinical data from patients infected with the strain, as well as viral prevalence estimates using real-time PCR, is also described.

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September 22, 2019  |  

Rodent papillomaviruses.

Preclinical infection model systems are extremely valuable tools to aid in our understanding of Human Papillomavirus (HPV) biology, disease progression, prevention, and treatments. In this context, rodent papillomaviruses and their respective infection models are useful tools but remain underutilized resources in the field of papillomavirus biology. Two rodent papillomaviruses, MnPV1, which infects the Mastomys species of multimammate rats, and MmuPV1, which infects laboratory mice, are currently the most studied rodent PVs. Both of these viruses cause malignancy in the skin and can provide attractive infection models to study the lesser understood cutaneous papillomaviruses that have been frequently associated with HPV-related skin cancers. Of these, MmuPV1 is the first reported rodent papillomavirus that can naturally infect the laboratory strain of mice. MmuPV1 is an attractive model virus to study papillomavirus pathogenesis because of the ubiquitous availability of lab mice and the fact that this mouse species is genetically modifiable. In this review, we have summarized the knowledge we have gained about PV biology from the study of rodent papillomaviruses and point out the remaining gaps that can provide new research opportunities.

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September 22, 2019  |  

Influenza virus infection causes global RNAPII termination defects.

Viral infection perturbs host cells and can be used to uncover regulatory mechanisms controlling cellular responses and susceptibility to infections. Using cell biological, biochemical, and genetic tools, we reveal that influenza A virus (IAV) infection induces global transcriptional defects at the 3′ ends of active host genes and RNA polymerase II (RNAPII) run-through into extragenic regions. Deregulated RNAPII leads to expression of aberrant RNAs (3′ extensions and host-gene fusions) that ultimately cause global transcriptional downregulation of physiological transcripts, an effect influencing antiviral response and virulence. This phenomenon occurs with multiple strains of IAV, is dependent on influenza NS1 protein, and can be modulated by SUMOylation of an intrinsically disordered region (IDR) of NS1 expressed by the 1918 pandemic IAV strain. Our data identify a strategy used by IAV to suppress host gene expression and indicate that polymorphisms in IDRs of viral proteins can affect the outcome of an infection.

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September 22, 2019  |  

Long-read sequencing revealed an extensive transcript complexity in herpesviruses.

Long-read sequencing (LRS) techniques are very recent advancements, but they have already been used for transcriptome research in all of the three subfamilies of herpesviruses. These techniques have multiplied the number of known transcripts in each of the examined viruses. Meanwhile, they have revealed a so far hidden complexity of the herpesvirus transcriptome with the discovery of a large number of novel RNA molecules, including coding and non-coding RNAs, as well as transcript isoforms, and polycistronic RNAs. Additionally, LRS techniques have uncovered an intricate meshwork of transcriptional overlaps between adjacent and distally located genes. Here, we review the contribution of LRS to herpesvirus transcriptomics and present the complexity revealed by this technology, while also discussing the functional significance of this phenomenon.

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September 22, 2019  |  

Next-generation sequencing for pathogen detection and identification

Over the past decade, the field of genomics has seen such drastic improvements in sequencing chemistries that high-throughput sequencing, or next-generation sequencing (NGS), is being applied to generate data across many disciplines. NGS instruments are becoming less expensive, faster, and smaller, and therefore are being adopted in an increasing number of laboratories, including clinical laboratories. Thus far, clinical use of NGS has been mostly focused on the human genome, for purposes such as characterizing the molecular basis of cancer or for diagnosing and understanding the basis of rare genetic disorders. There are, however, an increasing number of examples whereby NGS is employed to discover novel pathogens, and these cases provide precedent for the use of NGS in microbial diagnostics. NGS has many advantages over traditional microbial diagnostic methods, such as unbiased rather than pathogen-specific protocols, ability to detect fastidious or non-culturable organisms, and ability to detect co-infections. One of the most impressive advantages of NGS is that it requires little or no prior knowledge of the pathogen, unlike many other diagnostic assays; therefore for pathogen discovery, NGS is very valuable. However, despite these advantages, there are challenges involved in implementing NGS for routine clinical microbiological diagnosis. We discuss these advantages and challenges in the context of recently described research studies.

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September 22, 2019  |  

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics.

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio’s single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing.© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

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September 22, 2019  |  

Sixteen diverse laboratory mouse reference genomes define strain-specific haplotypes and novel functional loci.

We report full-length draft de novo genome assemblies for 16 widely used inbred mouse strains and find extensive strain-specific haplotype variation. We identify and characterize 2,567 regions on the current mouse reference genome exhibiting the greatest sequence diversity. These regions are enriched for genes involved in pathogen defence and immunity and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. We used these genomes to improve the mouse reference genome, resulting in the completion of 10 new gene structures. Also, 62 new coding loci were added to the reference genome annotation. These genomes identified a large, previously unannotated, gene (Efcab3-like) encoding 5,874 amino acids. Mutant Efcab3-like mice display anomalies in multiple brain regions, suggesting a possible role for this gene in the regulation of brain development.

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September 22, 2019  |  

No assembly required: Full-length MHC class I allele discovery by PacBio circular consensus sequencing.

Single-molecule real-time (SMRT) sequencing technology with the Pacific Biosciences (PacBio) RS II platform offers the potential to obtain full-length coding regions (~1100-bp) from MHC class I cDNAs. Despite the relatively high error rate associated with SMRT technology, high quality sequences can be obtained by circular consensus sequencing (CCS) due to the random nature of the error profile. In the present study we first validated the ability of SMRT-CCS to accurately identify class I transcripts in Mauritian-origin cynomolgus macaques (Macaca fascicularis) that have been characterized previously by cloning and Sanger-based sequencing as well as pyrosequencing approaches. We then applied this SMRT-CCS method to characterize 60 novel full-length class I transcript sequences expressed by a cohort of cynomolgus macaques from China. The SMRT-CCS method described here provides a straightforward protocol for characterization of unfragmented single-molecule cDNA transcripts that will potentially revolutionize MHC class I allele discovery in nonhuman primates and other species. Published by Elsevier Inc.

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September 22, 2019  |  

Avian transcriptomics: opportunities and challenges

Recent developments in next-generation sequencing technologies have greatly facilitated the study of whole transcriptomes in model and non-model species. Studying the transcriptome and how it changes across a variety of biological conditions has had major implications for our understanding of how the genome is regulated in different contexts, and how to interpret adaptations and the phenotype of an organism. The aim of this review is to highlight the potential of these new technologies for the study of avian transcriptomics, and to summarise how transcriptomics has been applied in ornithology. A total of 81 peer-reviewed scientific articles that used transcriptomics to answer questions within a broad range of study areas in birds are used as examples throughout the review. We further provide a quick guide to highlight the most important points which need to be take into account when planning a transcriptomic study in birds, and discuss how researchers with little background in molecular biology can avoid potential pitfalls. Suggestions for further reading are supplied throughout. We also discuss possible future developments in the technology platforms used for ribonucleic acid sequencing. By summarising how these novel technologies can be used to answer questions that have long been asked by ornithologists, we hope to bridge the gap between traditional ornithology and genomics, and to stimulate more interdisciplinary research.

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