16SPIP: a comprehensive analysis pipeline for rapid pathogen detection in clinical samples based on 16S metagenomic sequencing

Next-generation sequencing (NGS) technology has shown promise for the detection of human pathogens from clinical samples. However, one of the major obstacles to the use of NGS in diagnostic microbiology is the low ratio of pathogen DNA to human DNA in most clinical specimens. In this study, we aimed to develop a specimen-processing protocol to remove human DNA and enrich specimens for bacterial and viral DNA for shotgun metagenomic sequencing. Cerebrospinal fluid (CSF) and nasopharyngeal aspirate (NPA) specimens, spiked with control bacterial and viral pathogens, were processed using either a commercially available kit (MolYsis) or various detergents followed by DNase prior to the extraction of DNA. Relative quantities of human DNA and pathogen DNA were determined by real-time PCR. The MolYsis kit did not improve the pathogen-to-human DNA ratio, but significant reductions (>95%;P< 0.001) in human DNA with minimal effect on pathogen DNA were achieved in samples that were treated with 0.025% saponin, a nonionic surfactant. Specimen preprocessing significantly decreased NGS reads mapped to the human genome (P< 0.05) and improved the sensitivity of pathogen detection (P< 0.01), with a 20- to 650-fold increase in the ratio of microbial reads to human reads. Preprocessing also permitted the detection of pathogens that were undetectable in the unprocessed samples. Our results demonstrate a simple method for the reduction of background human DNA for metagenomic detection for a broad range of pathogens in clinical samples.

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Microbiome population dynamics of cold smoked sockeye salmon during refrigerated storage and after culture enrichment

Journal of Food Protection ◽

10.4315/jfp-21-228 ◽

2021 ◽

Author(s):

Karen Jarvis ◽

Chiun-Kang Hsu ◽

James B. Pettengill ◽

John Ihrie ◽

Hiren Karathia ◽

...

Keyword(s):

Population Dynamics ◽

Foodborne Pathogens ◽

Sockeye Salmon ◽

Pathogen Detection ◽

Rrna Gene ◽

Metagenomic Sequencing ◽

Smoked Salmon ◽

Spoilage Bacteria ◽

Culture Enrichment ◽

Processing And Storage

Cold smoked salmon is a ready-to-eat seafood product of high commercial importance. The processing and storage steps facilitate the introduction, growth and persistence of foodborne pathogens and spoilage bacteria. The growth of commensal bacteria during storage and once the product is opened also influence the quality and safety of cold smoked salmon. Here we investigated the microbial community through targeted 16s rRNA gene and shotgun metagenomic sequencing, as means to better understand the interactions among bacteria in cold smoked salmon. Cold smoked salmon samples were tested over 30 days of aerobic storage at 4℃ and cultured at each timepoint in buffered Listeria enrichment broth (BLEB) commonly used to detect Listeria in foods. The microbiomes were comprised of Firmicutes and Proteobacteria namely, Carnobacterium , Brochothrix , Pseudomonas , Serratia , and Psychrobacter . Pseudomonas species were the most diverse species with 181 taxa identified. Additionally, we identified potential homologs to 10 classes of bacteriocins in microbiomes of cold smoked salmon stored at 4°C and corresponding BLEB culture enrichments. The findings presented here contribute to our understanding of microbiome population dynamics in cold smoked salmon, including changes in bacterial taxa during aerobic cold storage and after culture enrichment. This may facilitate improvements to pathogen detection and quality preservation of this food.

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Correction to: Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples

Microbiome ◽

10.1186/s40168-017-0351-x ◽

2017 ◽

Vol 5 (1) ◽

Cited By ~ 1

Author(s):

Dagmara W. Lewandowska ◽

Osvaldo Zagordi ◽

Fabienne-Desirée Geissberger ◽

Verena Kufner ◽

Stefan Schmutz ◽

...

Keyword(s):

Sample Preparation ◽

Clinical Samples ◽

Metagenomic Sequencing

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Benchmark of thirteen bioinformatic pipelines for metagenomic virus diagnostics using datasets from clinical samples

10.1101/2021.05.04.21256618 ◽

2021 ◽

Author(s):

Jutte J.C. de Vries ◽

Julianne R. Brown ◽

Nicole Fischer ◽

Igor A. Sidorov ◽

Sofia Morfopoulou ◽

...

Keyword(s):

De Novo ◽

Respiratory Infections ◽

Limit Of Detection ◽

Bioinformatic Analysis ◽

Clinical Samples ◽

Mixed Infections ◽

Metagenomic Sequencing ◽

User Friendliness ◽

Viral Pathogens ◽

Clinical Diagnostic

Metagenomic sequencing is increasingly being used in clinical settings for difficult to diagnose cases. The performance of viral metagenomic protocols relies to a large extent on the bioinformatic analysis. In this study, the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS) initiated a benchmark of metagenomic pipelines currently used in clinical virological laboratories. Methods Metagenomic datasets from 13 clinical samples from patients with encephalitis or viral respiratory infections characterized by PCR were selected. The datasets were analysed with 13 different pipelines currently used in virological diagnostic laboratories of participating ENNGS members. The pipelines and classification tools were: Centrifuge, DAMIAN, DIAMOND, DNASTAR, FEVIR, Genome Detective, Jovian, MetaMIC, MetaMix, One Codex, RIEMS, VirMet, and Taxonomer. Performance, characteristics, clinical use, and user-friendliness of these pipelines were analysed. Results Overall, viral pathogens with high loads were detected by all the evaluated metagenomic pipelines. In contrast, lower abundance pathogens and mixed infections were only detected by 3/13 pipelines, namely DNASTAR, FEVIR, and MetaMix. Overall sensitivity ranged from 80% (10/13) to 100% (13/13 datasets). Overall positive predictive value ranged from 71-100%. The majority of the pipelines classified sequences based on nucleotide similarity (8/13), only a minority used amino acid similarity, and 6 of the 13 pipelines assembled sequences de novo. No clear differences in performance were detected that correlated with these classification approaches. Read counts of target viruses varied between the pipelines over a range of 2-3 log, indicating differences in limit of detection. Conclusion A wide variety of viral metagenomic pipelines is currently used in the participating clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implicating the need for standardization and validation of metagenomic analysis for clinical diagnostic use. Future studies should address the selective effects due to the choice of different reference viral databases.

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TagSeqTools: a flexible and comprehensive analysis pipeline for NAD tagSeq data

10.1101/2020.03.09.982934 ◽

2020 ◽

Cited By ~ 1

Author(s):

Huan Zhong ◽

Zongwei Cai ◽

Zhu Yang ◽

Yiji Xia

Keyword(s):

Rna Sequencing ◽

Comprehensive Analysis ◽

Enzymatic Reactions ◽

Computational Tool ◽

Sequencing Data ◽

Analysis Pipeline ◽

Oxford Nanopore ◽

Long Read ◽

Identification And Characterization

AbstractNAD tagSeq has recently been developed for the identification and characterization of NAD+-capped RNAs (NAD-RNAs). This method adopts a strategy of chemo-enzymatic reactions to label the NAD-RNAs with a synthetic RNA tag before subjecting to the Oxford Nanopore direct RNA sequencing. A computational tool designed for analyzing the sequencing data of tagged RNA will facilitate the broader application of this method. Hence, we introduce TagSeqTools as a flexible, general pipeline for the identification and quantification of tagged RNAs (i.e., NAD+-capped RNAs) using long-read transcriptome sequencing data generated by NAD tagSeq method. TagSeqTools comprises two major modules, TagSeek for differentiating tagged and untagged reads, and TagSeqQuant for the quantitative and further characterization analysis of genes and isoforms. Besides, the pipeline also integrates some advanced functions to identify antisense or splicing, and supports the data reformation for visualization. Therefore, TagSeqTools provides a convenient and comprehensive workflow for researchers to analyze the data produced by the NAD tagSeq method or other tagging-based experiments using Oxford nanopore direct RNA sequencing. The pipeline is available at https://github.com/dorothyzh/TagSeqTools, under Apache License 2.0.

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Third-Generation Sequencing in the Clinical Laboratory: Exploring the Advantages and Challenges of Nanopore Sequencing

Journal of Clinical Microbiology ◽

10.1128/jcm.01315-19 ◽

2019 ◽

Vol 58 (1) ◽

Cited By ~ 17

Author(s):

Lauren M. Petersen ◽

Isabella W. Martin ◽

Wayne E. Moschetti ◽

Colleen M. Kershaw ◽

Gregory J. Tsongalis

Keyword(s):

Infectious Disease ◽

Pathogen Detection ◽

Clinical Laboratory ◽

Low Cost ◽

Turnaround Time ◽

Metagenomic Sequencing ◽

Nanopore Sequencing ◽

Advantages And Disadvantages ◽

Disease Diagnostics ◽

Infectious Disease Diagnostics

ABSTRACT Metagenomic sequencing for infectious disease diagnostics is an important tool that holds promise for use in the clinical laboratory. Challenges for implementation so far include high cost, the length of time to results, and the need for technical and bioinformatics expertise. However, the recent technological innovation of nanopore sequencing from Oxford Nanopore Technologies (ONT) has the potential to address these challenges. ONT sequencing is an attractive platform for clinical laboratories to adopt due to its low cost, rapid turnaround time, and user-friendly bioinformatics pipelines. However, this method still faces the problem of base-calling accuracy compared to other platforms. This review highlights the general challenges of pathogen detection in clinical specimens by metagenomic sequencing, the advantages and disadvantages of the ONT platform, and how research to date supports the potential future use of nanopore sequencing in infectious disease diagnostics.

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Assessment of metagenomic Nanopore and Illumina sequencing for recovering whole genome sequences of chikungunya and dengue viruses directly from clinical samples

Eurosurveillance ◽

10.2807/1560-7917.es.2018.23.50.1800228 ◽

2018 ◽

Vol 23 (50) ◽

Cited By ~ 40

Author(s):

Liana E. Kafetzopoulou ◽

Kyriakos Efthymiadis ◽

Kuiama Lewandowski ◽

Ant Crook ◽

Dan Carter ◽

...

Keyword(s):

Illumina Miseq ◽

Clinical Samples ◽

Whole Genome ◽

Metagenomic Sequencing ◽

Front Line ◽

Genome Sequences ◽

Dengue Viruses ◽

Virus Identification ◽

Reverse Transcription Pcr ◽

Oxford Nanopore

Background The recent global emergence and re-emergence of arboviruses has caused significant human disease. Common vectors, symptoms and geographical distribution make differential diagnosis both important and challenging. Aim To investigate the feasibility of metagenomic sequencing for recovering whole genome sequences of chikungunya and dengue viruses from clinical samples. Methods We performed metagenomic sequencing using both the Illumina MiSeq and the portable Oxford Nanopore MinION on clinical samples which were real-time reverse transcription-PCR (qRT-PCR) positive for chikungunya (CHIKV) or dengue virus (DENV), two of the most important arboviruses. A total of 26 samples with a range of representative clinical Ct values were included in the study. Results Direct metagenomic sequencing of nucleic acid extracts from serum or plasma without viral enrichment allowed for virus identification, subtype determination and elucidated complete or near-complete genomes adequate for phylogenetic analysis. One PCR-positive CHIKV sample was also found to be coinfected with DENV. Conclusions This work demonstrates that metagenomic whole genome sequencing is feasible for the majority of CHIKV and DENV PCR-positive patient serum or plasma samples. Additionally, it explores the use of Nanopore metagenomic sequencing for DENV and CHIKV, which can likely be applied to other RNA viruses, highlighting the applicability of this approach to front-line public health and potential portable applications using the MinION.

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Reporter Phage-Based Detection of Bacterial Pathogens: Design Guidelines and Recent Developments

Viruses ◽

10.3390/v12090944 ◽

2020 ◽

Vol 12 (9) ◽

pp. 944 ◽

Cited By ~ 4

Author(s):

Susanne Meile ◽

Samuel Kilcher ◽

Martin J. Loessner ◽

Matthew Dunne

Keyword(s):

Viral Genome ◽

Pathogen Detection ◽

Bacterial Pathogens ◽

Design Guidelines ◽

Detection Methods ◽

Clinical Samples ◽

Cell Infection ◽

Reporter Protein ◽

Detection Systems ◽

Conventional Culture

Fast and reliable detection of bacterial pathogens in clinical samples, contaminated food products, and water supplies can drastically improve clinical outcomes and reduce the socio-economic impact of disease. As natural predators of bacteria, bacteriophages (phages) have evolved to bind their hosts with unparalleled specificity and to rapidly deliver and replicate their viral genome. Not surprisingly, phages and phage-encoded proteins have been used to develop a vast repertoire of diagnostic assays, many of which outperform conventional culture-based and molecular detection methods. While intact phages or phage-encoded affinity proteins can be used to capture bacteria, most phage-inspired detection systems harness viral genome delivery and amplification: to this end, suitable phages are genetically reprogrammed to deliver heterologous reporter genes, whose activity is typically detected through enzymatic substrate conversion to indicate the presence of a viable host cell. Infection with such engineered reporter phages typically leads to a rapid burst of reporter protein production that enables highly sensitive detection. In this review, we highlight recent advances in infection-based detection methods, present guidelines for reporter phage construction, outline technical aspects of reporter phage engineering, and discuss some of the advantages and pitfalls of phage-based pathogen detection. Recent improvements in reporter phage construction and engineering further substantiate the potential of these highly evolved nanomachines as rapid and inexpensive detection systems to replace or complement traditional diagnostic approaches.

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The Platelet Fraction Is a Novel Reservoir to Detect Lyme Borrelia in Blood

Biology ◽

10.3390/biology9110366 ◽

2020 ◽

Vol 9 (11) ◽

pp. 366

Author(s):

Victoria P. Sanderson ◽

Iain L. Mainprize ◽

Lisette Verzijlenberg ◽

Cezar M. Khursigara ◽

Melanie K. B. Wills

Keyword(s):

Pathogen Detection ◽

Bacterial Culture ◽

Direct Detection ◽

Laboratory Culture ◽

Detection Methods ◽

Clinical Samples ◽

Blood Collection ◽

Processing Conditions ◽

Proof Of Concept ◽

Clinical Detection

Serological diagnosis of Lyme disease suffers from considerable limitations. Yet, the technique cannot currently be replaced by direct detection methods, such as bacterial culture or molecular analysis, due to their inadequate sensitivity. The low bacterial burden in vasculature and lack of consensus around blood-based isolation of the causative pathogen, Borrelia burgdorferi, are central to this challenge. We therefore addressed methodological optimization of Borrelia recovery from blood, first by analyzing existing protocols, and then by using experimentally infected human blood to identify the processing conditions and fractions that increase Borrelia yield. In this proof-of-concept study, we now report two opportunities to improve recovery and detection of Borrelia from clinical samples. To enhance pathogen viability and cultivability during whole blood collection, citrate anticoagulant is superior to more commonly used EDTA. Despite the widespread reliance on serum and plasma as analytes, we found that the platelet fraction of blood concentrates Borrelia, providing an enriched resource for direct pathogen detection by microscopy, laboratory culture, Western blot, and PCR. The potential for platelets to serve as a reservoir for Borrelia and its diagnostic targets may transform direct clinical detection of this pathogen.

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Assessment of Viral Targeted Sequence Capture Using Nanopore Sequencing Directly from Clinical Samples

Viruses ◽

10.3390/v12121358 ◽

2020 ◽

Vol 12 (12) ◽

pp. 1358

Author(s):

Leonard Schuele ◽

Hayley Cassidy ◽

Erley Lizarazo ◽

Katrin Strutzberg-Minder ◽

Sabine Schuetze ◽

...

Keyword(s):

Influenza A ◽

Simultaneous Detection ◽

Clinical Samples ◽

Metagenomic Sequencing ◽

Genome Coverage ◽

Sequence Capture ◽

Shotgun Metagenomic Sequencing ◽

Oxford Nanopore ◽

The One ◽

Targeted Sequence Capture

Shotgun metagenomic sequencing (SMg) enables the simultaneous detection and characterization of viruses in human, animal and environmental samples. However, lack of sensitivity still poses a challenge and may lead to poor detection and data acquisition for detailed analysis. To improve sensitivity, we assessed a broad scope targeted sequence capture (TSC) panel (ViroCap) in both human and animal samples. Moreover, we adjusted TSC for the Oxford Nanopore MinION and compared the performance to an SMg approach. TSC on the Illumina NextSeq served as the gold standard. Overall, TSC increased the viral read count significantly in challenging human samples, with the highest genome coverage achieved using the TSC on the MinION. TSC also improved the genome coverage and sequencing depth in clinically relevant viruses in the animal samples, such as influenza A virus. However, SMg was shown to be adequate for characterizing a highly diverse animal virome. TSC on the MinION was comparable to the NextSeq and can provide a valuable alternative, offering longer reads, portability and lower initial cost. Developing new viral enrichment approaches to detect and characterize significant human and animal viruses is essential for the One Health Initiative.

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