scholarly journals Canine Meningoencephalitis of Unknown Origin—The Search for Infectious Agents in the Cerebrospinal Fluid via Deep Sequencing

2021 ◽  
Vol 8 ◽  
Author(s):  
Jasmin Nicole Nessler ◽  
Wendy Karen Jo ◽  
Albert D. M. E. Osterhaus ◽  
Martin Ludlow ◽  
Andrea Tipold
Keyword(s):  
Virus Infection ◽  
Deep Sequencing ◽  
Infectious Agent ◽  
Unknown Origin ◽  
Infectious Agents ◽  
Amplification Protocol ◽  
Fresh Frozen ◽  
Next Generation Sequencing Ngs ◽  
Single Primer ◽  
Generation Sequencing

Meningoencephalitis of unknown origin (MUO) describes a group of meningoencephalitides in dogs with a hitherto unknown trigger. An infectious agent has been suggested as one possible trigger of MUO but has not been proven so far. A relatively new method to screen for viral RNA or DNA is next-generation sequencing (NGS) or deep sequencing. In this study, a metagenomics analysis of the virome in a sample is analyzed and scanned for known or unknown viruses. We examined fresh-frozen CSF of 6 dogs with MUO via NGS using a modified sequence-independent, single-primer amplification protocol to detect a possible infectious trigger. Analysis of sequencing reads obtained from the six CSF samples showed no evidence of a virus infection. The inability to detect a viral trigger which could be implicated in the development of MUO in the examined population of European dogs, suggests that the current techniques are not sufficiently sensitive to identify a possible virus infection, that the virus is already eliminated at the time-point of disease outbreak, the trigger might be non-infectious or that there is no external trigger responsible for initiating MUO in dogs.

scholarly journals The effect of variant interference on de novo assembly for viral deep sequencing

2019 ◽  
Author(s):  
Christina J. Castro ◽  
Rachel L. Marine ◽  
Edward Ramos ◽  
Terry Fei Fan Ng
Keyword(s):  
Deep Sequencing ◽  
De Novo ◽  
Gc Content ◽  
Read Length ◽  
Viral Genomes ◽  
Minor Variant ◽  
Main Driver ◽  
Next Generation Sequencing Ngs ◽  
Viral Sequences ◽  
Generation Sequencing

AbstractViruses have high mutation rates and generally exist as a mixture of variants in biological samples. Next-generation sequencing (NGS) approach has surpassed Sanger for generating long viral sequences, yet how variants affect NGS de novo assembly remains largely unexplored. Our results from >15,000 simulated experiments showed that presence of variants can turn an assembly of one genome into tens to thousands of contigs. This “variant interference” (VI) is highly consistent and reproducible by ten most used de novo assemblers, and occurs independent of genome length, read length, and GC content. The main driver of VI is pairwise identities between viral variants. These findings were further supported by in silico simulations, where selective removal of minor variant reads from clinical datasets allow the “rescue” of full viral genomes from fragmented contigs. These results call for careful interpretation of contigs and contig numbers from de novo assembly in viral deep sequencing.

scholarly journals Next-generation sequencing in neuropathological diagnosis of infections of the nervous system

2016 ◽  
Author(s):  
Steven L. Salzberg ◽  
Florian Breitwieser ◽  
Anupama Kumar ◽  
Haiping Hao ◽  
Peter Burger ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Nervous System ◽  
Next Generation Sequencing ◽  
Large Scale ◽  
Infectious Agent ◽  
Next Generation ◽  
Dna And Rna ◽  
Wide Range ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Objective: To determine the feasibility of next-generation sequencing (NGS) microbiome approaches in the diagnosis of infectious disorders in brain or spinal cord biopsies in patients with suspected central nervous system (CNS) infections. Methods: In a prospective-pilot study, we applied NGS in combination with a new computational analysis pipeline to detect the presence of pathogenic microbes in brain or spinal cord biopsies from ten patients with neurological problems indicating possible infection but for whom conventional clinical and microbiology studies yielded negative or inconclusive results. Results: Direct DNA and RNA sequencing of brain tissue biopsies generated 8.3 million to 29.1 million sequence reads per sample, which successfully identified with high confidence the infectious agent in three patients, identified possible pathogens in two more, and helped to understand neuropathological processes in three others, demonstrating the power of large-scale unbiased sequencing as a novel diagnostic tool. Validation techniques confirmed the pathogens identified by NGS in each of the three positive cases. Clinical outcomes were consistent with the findings yielded by NGS on the presence or absence of an infectious pathogenic process in eight of ten cases, and were non-contributory in the remaining two. Conclusions: NGS-guided metagenomic studies of brain, spinal cord or meningeal biopsies offer the possibility for dramatic improvements in our ability to detect (or rule out) a wide range of CNS pathogens, with potential benefits in speed, sensitivity, and cost. NGS-based microbiome approaches present a major new opportunity to investigate the potential role of infectious pathogens in the pathogenesis of neuroinflammatory disorders.

scholarly journals HAPHPIPE: Haplotype Reconstruction and Phylodynamics for Deep Sequencing of Intra-Host Viral Populations

2020 ◽  
Author(s):  
Matthew L Bendall ◽  
Keylie M Gibson ◽  
Margaret C Steiner ◽  
Uzma Rentia ◽  
Marcos Pérez-Losada ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
De Novo ◽  
Consensus Sequence ◽  
Haplotype Reconstruction ◽  
Consensus Sequences ◽  
Genome Wide ◽  
Genomic Regions ◽  
Next Generation Sequencing Ngs ◽  
Ngs Data ◽  
Generation Sequencing

Abstract Deep sequencing of viral populations using next generation sequencing (NGS) offers opportunities to understand and investigate evolution, transmission dynamics, and population genetics. Currently, the standard practice for processing NGS data to study viral populations is to summarize all the observed sequences from a sample as a single consensus sequence, thus discarding valuable information about the intra-host viral molecular epidemiology. Furthermore, existing analytical pipelines may only analyze genomic regions involved in drug resistance, thus are not suited for full viral genome analysis. Here we present HAPHPIPE, a HAplotype and PHylodynamics PIPEline for genome-wide assembly of viral consensus sequences and haplotypes. The HAPHPIPE protocol includes modules for quality trimming, error correction, de novo assembly, alignment, and haplotype reconstruction. The resulting consensus sequences, haplotypes, and alignments can be further analyzed using a variety of phylogenetic and population genetic software. HAPHPIPE is designed to provide users with a single pipeline to rapidly analyze sequences from viral populations generated from NGS platforms and provide quality output properly formatted for downstream evolutionary analyses.

Progress Report: Next-Generation Sequencing (NGS), Multiplex Polymerase Chain Reaction (PCR), and Broad-Range Molecular Assays as Diagnostic Tools for Fever of Unknown Origin (FUO) Investigations in Adults

2021 ◽  
Author(s):  
William F Wright ◽  
Patricia J Simner ◽  
Karen C Carroll ◽  
Paul G Auwaerter
Keyword(s):  
Next Generation Sequencing ◽  
Fever Of Unknown Origin ◽  
Unknown Origin ◽  
Diagnostic Tools ◽  
Rrna Gene ◽  
Next Generation ◽  
Potential Cost ◽  
Microbial Identification ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Even well into the 21st century, infectious diseases still account for most causes of fever of unknown origin (FUO). Advances in molecular technologies, including broad-range PCR of the 16S rRNA gene followed by Sanger sequencing, multiplex PCR assays, and more recently, next-generation sequencing (NGS) applications, have transitioned from research methods to more commonplace in some clinical microbiology laboratories. They have the potential to supplant traditional microbial identification methods and antimicrobial susceptibility testing. Despite the remaining challenges with these technologies, publications in the past decade justify excitement about the potential to transform FUO investigations. We discuss available evidence using these molecular methods for FUO evaluations, including potential cost-benefits and future directions.

scholarly journals Comparison of double-stranded and single-stranded cfDNA library construction methods for targeted genome and methylation sequencing

2022 ◽  
Author(s):  
Jianchao Zheng ◽  
Zhilong Li ◽  
Xiuqing Zhang ◽  
Hongyun Zhang ◽  
Shida Zhu ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Mutation Detection ◽  
Therapy Response ◽  
Utilization Efficiency ◽  
Response Monitoring ◽  
Library Construction ◽  
Construction Methods ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Methylation Profiling

Cell-free DNA (cfDNA) profiling by deep sequencing (i.e., by next generation sequencing (NGS)) has wide applications in cancer diagnosis, prognosis, and therapy response monitoring. One key step of cfDNA deep sequencing workflow is NGS library construction, whose efficiency significantly affects the utilization efficiency of cfDNA molecules, and eventually determines effective sequencing depth and sequencing accuracy. In this study, we compared two different types of cfDNA library construction methods, namely double-stranded library (dsLib, the conventional method which captures dsDNA molecules) and single-stranded library (ssLib) preparation, which captures ssDNA molecules, for the applications of mutation detection and methylation profiling, respectively. Our results suggest that the dsLib method was suitable for mutation detection while the ssLib method proved more efficient for methylation analysis. Our findings could help researchers choose the more appropriate library construction method for corresponding downstream applications of cfDNA sequencing.

scholarly journals Lethal Encephalitis of Unknown Origin—Elucidation by Metagenomics

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 59
Author(s):  
Martin Beer
Keyword(s):  
Point Of Care ◽  
Animal Health ◽  
Unknown Origin ◽  
Emerging Viruses ◽  
Viral Pathogens ◽  
Point Of Care Diagnostics ◽  
Close Relationship ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Novel and (re)emerging viruses cause frequent threats to both human and animal health. Diagnostic metagenomics using unbiased next-generation sequencing (NGS) is the key method for the identification of new pathogens. With today’s available state-of-the-art platforms, NGS can be broadly used to also identify novel and unknown pathogens in different sample materials (even in point-of-care diagnostics) or to characterize the complete genomes of all types of pathogens. Nevertheless, the extreme numbers of sequence fragments resulting from NGS analyses requires not only novel diagnostic pipelines including powerful software tools for big data analysis but also a new dimension of knowledge and resources. We therefore developed and validated a universal workflow for metagenome diagnostics for the analysis of disease syndromes in both animals and humans. The metagenomics pipeline will be presented, and several examples with the detection and characterization of novel viruses will be shown. The power of diagnostic metagenomics will be presented with different examples focusing on lethal encephalitis cases in both animals and humans where we were able to identify a series of novel or unexpected viral pathogens. Furthermore, the detection of zoonotic pathogens was only possible by a “one-health” approach and the close relationship between veterinary and human medicine. The major aim of the presentation is to give an idea about the capabilities of modern NGS-based metagenomics and to learn more about the newly detected viral pathogens. Since a large proportion of severe encephalitis cases still remain unexplained, a main conclusion is the recommendation that those cases should be analyzed by using a modern and powerful metagenomics workflow.

scholarly journals Increasing importance of genomics in recognition of infectious animal diseases taking into account OIE requirements

2017 ◽  
Vol 73 (1) ◽  
pp. 10-14
Author(s):  
Woźniakowski Grzegorz. ◽  
Truszczyński Marian ◽  
Pejsak Zygmunt
Keyword(s):  
High Throughput Sequencing ◽  
Animal Health ◽  
Infectious Agents ◽  
Animal Diseases ◽  
Routine Diagnosis ◽  
Animal Pathogens ◽  
Polymerase Chain ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
World Organisation

Currently, the diagnosis methods applied in bacteriology and virology are frequently focused at modern molecular methods such as polymerase chain reaction (PCR), real-time PCR, loop-mediated isothermal amplification (LAMP) or confirmatory methods for already identified infectious agents of animals such as high throughput sequencing- HTS, or next-generation sequencing (NGS). A number of these methods are officially recommended by the World Organisation for Animal Health (OIE) for the routine diagnosis of major animal diseases with the greatest economical impact. The aim of this paper is to provide to a reader an overview on recent genomic methods used in diagnosis of infectious animal diseases along with their advantages and limitations. These methods facilitate efficient and reliable identification of animal pathogens and allow to characterize and sometimes to identify a newly recognized species of bacteria and viruses.

scholarly journals Μοριακοί μηχανισμοί κυτταρικού επαναπρογραμματισμού

2021 ◽  
Author(s):  
Ειρήνη Αλεξοπούλου
Keyword(s):  
Next Generation Sequencing ◽  
Virus Infection ◽  
Rna Seq ◽  
Next Generation ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Η παρούσα διδακτορική διατριβή μελετά τους μηχανισμούς ρύθμισης της επαγόμενης γονιδιακής έκφρασης ευκαρυωτικών κυττάρων σε επίπεδο ολόκληρου του γονιδιώματος και πιο συγκεκριμένα, την ταυτοποίηση των μοριακών διακοπτών, οι οποίοι βάση της συντονισμένης απόκρισης των κυττάρων, ελέγχουν τη λειτουργία των ενισχυτών σε διαφορετικούς κυτταρικούς τύπους πριν και μετά από ιϊκή μόλυνση. O χαρακτηρισμός των ενισχυτών που αποκρίνονται σε ιϊκές μολύνσεις στο ανθρώπινο γονιδίωμα απαιτείται για την κατανόηση του συντονισμού της ανοσολογικής απόκρισης και των μοριακών χαρακτηριστικών που κυμαίνονται από την πρόσδεση μεταγραφικών παραγόντων, συνενεργοποιητών, τροποποιητών και αναδιαμορφωτών της χρωματίνης μέχρι την ενεργοποίηση της μεταγραφής γονιδίων.Η έρευνά μας συνίσταται από μια πρωτοποριακή και ολιστική προσέγγιση του φαινομένου των ιϊκών μολύνσεων, η εφαρμογή της οποίας συνδυάζει όλα τα δεδομένα που προέρχονται από μια πληθώρα τεχνικών της γονιδιωματικής. Χρησιμοποιήθηκαν περισσότερες από 150 αναλύσεις των μεθόδων RNA-seq, ChIP-seq, DNaseI-seq, FAIRE-seq, STARR-seq και πολυεπίπεδων βιοπληροφορικών αναλύσεων, τα οποία έχουν ως κοινή μέθοδο ανάλυσης την τεχνολογία αλληλούχησης ευρείας κλίμακας νέας γενιάς (Next Generation Sequencing, NGS). Η σύγκριση των ρυθμιστικών προγραμμάτων γονιδιακής έκφρασης στη διάρκεια της μόλυνσης πραγματοποιήθηκε σε διαφορετικούς κυτταρικούς τύπους, στα επιθηλιακά κύτταρα HeLa και στα Β-λεμφοκύτταρα Namalwa, τα οποία διαμολύνθηκαν με ιό Sendai, ως περιβαλλοντικό ερέθισμα. Τα χαρακτηριστικά της χρωματίνης, το προφίλ του επιγονιδιώματος καθώς και η συμμετοχή της μεταγραφικής μηχανής σε γονιδιωματικές συντεταγμένες, όπου εδράζονται οι ενισχυτές και ένας μεγάλος αριθμός καλά χαρακτηρισμένων αντι-ιϊκών γονιδίων, έθεσαν τις βάσεις για την κατασκευή μιας "μοριακής-ψηφιακής εγκυκλοπαίδειας" των γονιδίων και των ενισχυτών. Η εγκυκλοπαίδεια χαρακτηρίζεται με υψηλή εν δυνάμει πιθανότητα να παρουσιάσει "αντι-ιϊκές ιδιότητες" και ονομάζεται ΆΤΛΑΝΤΑΣ των ιϊκών μολύνσεων (Human-ATLAS-of-Virus-Infection). Ο ΑΤΛΑΝΤΑΣ επικυρώθηκε με την εφαρμογή της μεθόδου ChIP-STARR-seq. Η πρωτοποριακή αυτή μέθοδος ευρείας κλίμακας μας επέτρεψε την ταυτόχρονη εξέταση της ιϊκά επαγόμενης ενεργότητας των πολυάριθμων περιοχών πρόσδεσης των μεταγραφικών ρυθμιστών της αντι-ιϊκής απόκρισης IRF3 και p65. Αναλύσεις βασισμένες στη συντήρηση των αλληλουχιών αποκάλυψαν ότι οι λειτουργικοί αντι-ιϊκοί ενισχυτές που περιέχουν συμπλέγματα IRF μοτίβων είναι σε μεγάλο βαθμό σταθεροί σε όλη την εξέλιξη καθώς βρίσκονται στα γονιδιώματα διαφορετικών ειδών που αντιπροσωπεύουν τα σπονδυλωτά, τα ασπόνδυλα, τα φυτά, ακόμη και μικρόβια, συμπεριλαμβανομένων των ιϊκών στελεχών. Αυτές οι απλές επαναλαμβανόμενες αλληλουχίες αποτελούν τα πρώτα παραδείγματα επαναλαμβανόμενων αλληλουχιών με αρχέγονη προέλευση ρύθμισης της αντι-ιϊκής απόκρισης. Προκειμένου να μελετηθεί συστηματικά η λειτουργία των συντηρημένων αλληλουχιών από τους παραπάνω διαφορετικούς οργανισμούς σε ανθρώπινα κύτταρα, αλλά και να διαπιστωθεί αν αυτές οι αλληλουχίες παρουσιάζουν και συντηρημένη ενεργότητα ενισχυτών, προβήκαμε σε κλωνοποίηση σε φορείς STARR. Έγινε διαμόλυνση σε κύτταρα HeLa και παρακολουθήσαμε την ικανότητά τους να ενεργοποιούν την έκφραση του γονίδιο αναφοράς GFP in νίνο. Αυτή είναι μία από τις λίγες φορές που οι ενισχυτές διατηρούνται συντηρημένοι σε διαφορετικά Φύλα και οι αλληλουχίες με την αρχέγονη προέλευση φαίνεται να λειτουργούν ως ρυθμιστές της ανθρώπινης επαγώγιμης γονιδιακής έκφρασης κατά την αντι-ιϊκή κυτταρική απόκριση. Με βάση όλα τα παραπάνω η ολοκλήρωση της διδακτορικής αυτής διατριβής οδηγεί στη λεπτομερέστερη παρατήρηση του μηχανισμού που χρησιμοποιούν τα ανθρώπινα κύτταρα για να αποκριθούν στη διάρκεια των ιϊκών μολύνσεων. Θέτει επίσης τις βάσεις για περαιτέρω διερεύνηση τους καθώς και στην ανακάλυψη άγνωστων ως σήμερα στοιχείων αυτής της διαδικασίας. Η αποκωδικοποίηση της ρυθμιστικής λογικής πίσω από την αναδιαμόρφωση του γονιδιώματος μετά από ιϊκή μόλυνση θα οδηγήσει σε βαθύτερη κατανόηση των βασικών αρχών οργάνωσης του γονιδιώματος και στη δυνατότητα πρόβλεψης της απόκρισης των ασθενών σε θεραπευτικές και προληπτικές αγωγές με απώτερο σκοπό στο μέλλον να εφαρμόζονται σε εξατομικευμένη βάση.

scholarly journals Serology and Amplicon Deep Sequencing of MSP1-Block 2 Haplotypes as New Tools for Plasmodium Vivax Infections

2021 ◽  
Author(s):  
Elizangela Farias ◽  
Maele Jordão ◽  
Ricardo Avila ◽  
Mirian Fagundes ◽  
Paulo Feuser ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Plasmodium Vivax ◽  
Deep Sequencing ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Multiple Infections ◽  
High Quality ◽  
Merozoite Surface Protein 1 ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Background: Relapses of Plasmodium vivax (P. vivax) infections are major causes of malaria morbidity, and tools for distinguishing relapses from reinfections are needed in malaria endemic areas. Methods: Herein, a panel of plasmas of 72 P. vivax-infected pregnant women, of whom 31 had had at least a recurrence of P. vivax infection, was used in a serology for IgM and IgG against 6 P. vivax-merozoite surface protein-1 (P. vivax-MSP1-Block 2) haplotype-specific peptides, in order to identify re-expositions to same haplotypes in the recurrences during the pregnancy. In parallel, we used the amplicon deep sequencing (ADS) with P. vivax-MSP1-Block 2 amplicons of the in eight blood samples of non-pregnant P. vivax-infected patients to identify multi or monoclonal infections based on MSP1-Block-2 haplotypes, and to quantify the reads of different haplotypes between those with multiclonal infections. We synthetized a new panel of overlapping peptides mapping each one of the six P. vivax-MSP1-Block 2 haplotypes and we validated with new IgM and IgG serology. Results: Most pregnant women presented IgM that recognized more than one peptide, thus indicating multiple infections by P. vivax-MSP1-Block 2 haplotypes. The same IgM anti-peptides remained in several women in the recurrent episodes most likely indicates re-exposure to the same haplotype of MSP1 Block 2. The IgG reactivity the IgM to IgG switch were low. The ADS using next-generation sequencing (NGS) identified multi- and monoinfection by P. vivax-MSP1-Block 2 haplotypes. Of eight patients, two of them had had the first P. vivax-infection. Monoinfections with P. vivax-MSP1-Block 2 haplotypes were observed in two prime-infected patients and three of patients with previous malaria. In all P. vivax-MSP1-Block 2 haplotype-monoinfected patients, the reactivity of IgM was observed only against overlapped peptides of the same haplotype detected in ADS, while for IgG, no reactivity was observed for any of the peptides of the same haplotype or the others.We were able to identify multiclonal infections through three haplotypes of P. vivax MSP1 Block 2 in three remaining patients, among which, there was always one majority haplotype that predominated with more of 95% of high-quality reads. The levels of haplotype-specific IgM in the serology correlated with the read ratios of each haplotype, but IgG levels not, including in one of the multiclonal infections, a minority haplotype was recognized with higher levels of IgG than that of the majority one. Conclusion: Our findings suggest that the combination of ADS and serology for P. vivax-MSP1-Block 2 haplotypes may be used as a new tool for distinguishing reinfections from relapses in malaria.

Sign in / Sign up

Export Citation Format

Share Document