A Versatile Sample Processing Workflow for Metagenomic Pathogen Detection

Metagenomics is currently the only generic method for pathogen detection. Starting from RNA allows the assessment of the whole sample community including RNA viruses. Here we present our modular concerted protocol for sample processing for diagnostic metagenomics analysis of human, animal, and food samples. The workflow does not rely on dedicated amplification steps at any stage in the process and, in contrast to published methods, libraries prepared accordingly will yield only minute amounts of unclassifiable reads. We confirmed the performance of the approach using a spectrum of pathogen/matrix-combinations showing it has the potential to become a commonly usable analytical framework.

[1]  M. Beer,et al.  Spatio-temporal Analysis of the Genetic Diversity of Arctic Rabies Viruses and Their Reservoir Hosts in Greenland , 2016, PLoS neglected tropical diseases.

[2]  I. Weissman,et al.  Index switching causes “spreading-of-signal” among multiplexed samples in Illumina HiSeq 4000 DNA sequencing , 2017, bioRxiv.

[3]  J. Rosenfeld,et al.  MIPEP recessive variants cause a syndrome of left ventricular non-compaction, hypotonia, and infantile death , 2016, Genome Medicine.

[4]  M. Beer,et al.  Detection of a Novel Bovine Astrovirus in a Cow with Encephalitis. , 2015, Transboundary and emerging diseases.

[5]  Klaus Stark,et al.  Epidemic profile of Shiga-toxin-producing Escherichia coli O104:H4 outbreak in Germany. , 2011, The New England journal of medicine.

[6]  M. Beer,et al.  Porcine Epidemic Diarrhea in Europe: In-Detail Analyses of Disease Dynamics and Molecular Epidemiology , 2017, Viruses.

[7]  Stephan Günther,et al.  Emergence of Zaire Ebola virus disease in Guinea. , 2014, The New England journal of medicine.

[8]  Farid E Ahmed,et al.  Microarray RNA transcriptional profiling: Part I. Platforms, experimental design and standardization , 2006, Expert review of molecular diagnostics.

[9]  A. Nitsche,et al.  Protocol for Metagenomic Virus Detection in Clinical Specimens , 2015, Emerging infectious diseases.

[10]  Robert Schlaberg,et al.  Validation of Metagenomic Next-Generation Sequencing Tests for Universal Pathogen Detection. , 2017, Archives of pathology & laboratory medicine.

[11]  Martin Beer,et al.  RIEMS: a software pipeline for sensitive and comprehensive taxonomic classification of reads from metagenomics datasets , 2015, BMC Bioinformatics.

[12]  T. Tatusova,et al.  Solving the Problem: Genome Annotation Standards before the Data Deluge , 2011, Standards in genomic sciences.

[13]  A. Papa,et al.  Bacterial pathogens and endosymbionts in ticks. , 2017, Ticks and tick-borne diseases.

[14]  K. Eilbeck,et al.  Unbiased Detection of Respiratory Viruses by Use of RNA Sequencing-Based Metagenomics: a Systematic Comparison to a Commercial PCR Panel , 2016, Journal of Clinical Microbiology.

[15]  S. Becker,et al.  Genetic characterization of Arrabida virus, a novel phlebovirus isolated in South Portugal. , 2016, Virus research.

[16]  Bernd Hoffmann,et al.  Novel Orthobunyavirus in Cattle, Europe, 2011 , 2012, Emerging infectious diseases.

[17]  Vincent Montoya,et al.  Metagenomics for pathogen detection in public health , 2013, Genome Medicine.

[18]  Raju V. Misra,et al.  Challenges of the Unknown: Clinical Application of Microbial Metagenomics , 2015, International journal of genomics.

[19]  G. Marth,et al.  Primer-site SNPs mask mutations , 2007, Nature Methods.

[20]  Frank M. Aarestrup,et al.  Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid , 2017, bioRxiv.

[21]  Luis Pedro Coelho,et al.  Towards standards for human fecal sample processing in metagenomic studies , 2017, Nature Biotechnology.

[22]  O. Ozhelvaci,et al.  Evaluation of convenient pretreatment protocols for RNA virus metagenomics in serum and tissue samples. , 2015, Journal of virological methods.

[23]  T. Mettenleiter,et al.  Metagenomic approaches to identifying infectious agents. , 2016, Revue scientifique et technique.

[24]  H. Köhler,et al.  Characterization of tuberculous granulomas in different stages of progression and associated tertiary lymphoid tissue in goats experimentally infected with Mycobacterium avium subsp. hominissuis. , 2016, Comparative immunology, microbiology and infectious diseases.

[25]  M. Beer,et al.  Complete Coding Genome Sequence of Putative Novel Bluetongue Virus Serotype 27 , 2015, Genome Announcements.

[26]  J. Utzinger,et al.  Metagenomic diagnostics for the simultaneous detection of multiple pathogens in human stool specimens from Côte d'Ivoire: a proof-of-concept study. , 2016, Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases.

[27]  T. Mettenleiter.  The First "Virus Hunters". , 2017, Advances in virus research.

[28]  M. Beer,et al.  A Variegated Squirrel Bornavirus Associated with Fatal Human Encephalitis. , 2015, The New England journal of medicine.

[29]  A. Osterhaus,et al.  Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. , 2012, The New England journal of medicine.

[30]  J. Dave,et al.  Potential of Probiotic Lactobacillus Strains as Food Additives , 2012 .

[31]  Paul Turner,et al.  Reagent and laboratory contamination can critically impact sequence-based microbiome analyses , 2014, BMC Biology.

[32]  M. Beer,et al.  In-depth genome analyses of viruses from vaccine-derived rabies cases and corresponding live-attenuated oral rabies vaccines. , 2018, Vaccine.

[33]  H. Hotzel,et al.  Comparison of 13 single-round and nested PCR assays targeting IS900, ISMav2, f57 and locus 255 for detection of Mycobacterium avium subsp. paratuberculosis. , 2008, Veterinary microbiology.

[34]  T. G. Mitchell,et al.  Next generation multilocus sequence typing (NGMLST) and the analytical software program MLSTEZ enable efficient, cost-effective, high-throughput, multilocus sequencing typing. , 2015, Fungal genetics and biology : FG & B.

[35]  Jelle Matthijnssens,et al.  Modular approach to customise sample preparation procedures for viral metagenomics: a reproducible protocol for virome analysis , 2015, Scientific Reports.

[36]  Jeffrey Hoorfar,et al.  Surveillance of Foodborne Pathogens: Towards Diagnostic Metagenomics of Fecal Samples , 2018, Genes.

[37]  M. Beer,et al.  The Origin of Biased Sequence Depth in Sequence-Independent Nucleic Acid Amplification and Optimization for Efficient Massive Parallel Sequencing , 2013, PloS one.

[38]  Martin Beer,et al.  Go COMPARE! “One serves all” next-generation sequencing framework , 2017 .

[39]  F. Conraths,et al.  Monitoring of Putative Vectors of Bluetongue Virus Serotype 8, Germany , 2009, Emerging infectious diseases.

[40]  P. Svastova,et al.  Methods of mycobacterial DNA isolation from different biological material: a review , 2018 .

[41]  Jennifer L. Gardy,et al.  Towards a genomics-informed, real-time, global pathogen surveillance system , 2017, Nature Reviews Genetics.

[42]  Joseph L. DeRisi,et al.  Illuminating uveitis: metagenomic deep sequencing identifies common and rare pathogens , 2016, bioRxiv.

[43]  F. van Nieuwerburgh,et al.  Library construction for next-generation sequencing: overviews and challenges. , 2014, BioTechniques.

[44]  Linda Koch.  Metagenomics: Pathogen diagnostics for the masses , 2016, Nature Reviews Genetics.

[45]  M. Beer,et al.  A Comprehensive Deep Sequencing Strategy for Full-Length Genomes of Influenza A , 2011, PloS one.

[46]  M. Pallen.  Diagnostic metagenomics: potential applications to bacterial, viral and parasitic infections , 2014, Parasitology.

[47]  M. Beer,et al.  A novel alphaherpesvirus associated with fatal diseases in banded Penguins. , 2017, The Journal of general virology.

[48]  F. Aarestrup,et al.  Sharing Data for Global Infectious Disease Surveillance and Outbreak Detection. , 2016, Trends in microbiology.

[49]  M. Beer,et al.  A novel astrovirus associated with encephalitis and ganglionitis in domestic sheep , 2017, Transboundary and emerging diseases.

[50]  Charles Y. Chiu,et al.  Clinical metagenomic identification of Balamuthia mandrillaris encephalitis and assembly of the draft genome: the continuing case for reference genome sequencing , 2015, Genome Medicine.

[51]  A. Cay,et al.  Schmallenberg Virus Circulation in Culicoides in Belgium in 2012: Field Validation of a Real Time RT-PCR Approach to Assess Virus Replication and Dissemination in Midges , 2014, PloS one.

[52]  S. Olabarriaga,et al.  Performance of VIDISCA-454 in Feces-Suspensions and Serum , 2012, Viruses.