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

AC electrokinetics is a promising approach for sample preparation and reaction enhancement in lab-on-a-chip devices. However, relative little has been done on the electrokinetic manipulation of physiological fluids and buffers with similar properties, such as conductivity. Herein, electrokinetic manipulation of fluids with a wide range of conductivities has been studied as a function of voltage and frequency. AC electrothermal flow is determined to dominate the fluid motion when the applied frequency of the AC potential is above 100 kHz. Interestingly, experimental data deviate from theoretical prediction for fluids with high conductivities (> 1 Sm−1). The deviation can be understood by voltage modulated electrochemical reactions and should be accounted for when manipulating clinical materials with high conductivities. The study will provide useful in sights in designing lab-on-a-chip devices for manipulating clinical samples in the future.

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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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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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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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1756. Prediction of Bloodstream Infection Prior to Onset of Symptoms by Plasma Metagenomic Sequencing in Pediatric Patients With Relapsed or Refractory Malignancy (PREDSEQ)

Open Forum Infectious Diseases ◽

10.1093/ofid/ofy209.141 ◽

2018 ◽

Vol 5 (suppl_1) ◽

pp. S60-S60 ◽

Cited By ~ 1

Author(s):

Kathryn Goggin ◽

Yuki Inaba ◽

Veronica Gonzalez-Pena ◽

Kim J Allison ◽

Ka Lok Chan ◽

...

Keyword(s):

High Risk ◽

Bloodstream Infection ◽

Pediatric Patients ◽

Clinical Samples ◽

Metagenomic Sequencing ◽

Research Support ◽

Documented Infection ◽

Risk Patients ◽

Bacteria And Fungi ◽

Refractory Malignancy

Abstract Background Patients undergoing treatment for relapsed or refractory malignancies are at high risk of life-threatening bloodstream infection (BSI). A predictive screening test for BSI might allow pre-emptive therapy, but no validated test is currently available. We tested the hypothesis that plasma metagenomic next generation pathogen sequencing (NGS) would predict BSI before the onset of attributable symptoms. Methods We enrolled 31 pediatric patients receiving for treatment relapsed or refractory malignancy in an IRB-approved prospective cohort study (PREDSEQ) of predictive sequencing. Episodes of febrile neutropenia or documented infection were collected prospectively from the medical record. BSI was defined according to NHSN criteria. Control Samples were defined as samples collected ≥7 clear days before or after any fever or documented infection. Residual clinical samples were stored for NGS; after filtering human sequences, reads were aligned to a curated pathogen database, and organisms above a predefined threshold were reported (Karius Inc., Redwood City, CA). Only bacteria and fungi were included in this analysis. Results A total of 11 BSI episodes occurred in 9 participants (Table 1) during the study period. Predictive sensitivity of NGS in the 2 days before onset of infection (n = 9) was 78% (95% CI 45–94%), and diagnostic sensitivity on the day of infection (n = 11) was 82% (95% CI 52–95%). Specificity of NGS for development of fever or infection within 7 days (n = 16) was 81% (95% CI 57–93%). NGS was positive up to 6 days prior to onset of BSI. In samples collected before or during documented infections, NGS also identified additional bacteria and fungi that were not detected by standard clinical testing. Conclusion Plasma NGS shows promise for the detection of BSI prior to onset of symptoms in high-risk patients. Disclosures K. Goggin, Karius Inc.: Investigator, Research support. K. L. Chan, Karius Inc.: Employee, Salary. D. Hollemon, Karius Inc.: Employee, Salary. A. Ahmed, Karius, Inc.: Employee, Salary. D. Hong, Karius, Inc.: Employee, Salary. R. Hayden, Roche Molecular: Scientific Advisor, Consulting fee. Abbott Molecular: Scientific Advisor, Consulting fee. Quidel: Scientific Advisor, Consulting fee. C. Gawad, Karius Inc.: Investigator, Research support. J. Wolf, Karius Inc.: Investigator, Research support.

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Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing

Journal of Clinical Microbiology ◽

10.1128/jcm.03050-15 ◽

2016 ◽

Vol 54 (4) ◽

pp. 919-927 ◽

Cited By ~ 66

Author(s):

Mohammad R. Hasan ◽

Arun Rawat ◽

Patrick Tang ◽

Puthen V. Jithesh ◽

Eva Thomas ◽

...

Keyword(s):

Next Generation Sequencing ◽

Pathogen Detection ◽

Clinical Samples ◽

Metagenomic Sequencing ◽

Next Generation ◽

Simple Method ◽

Clinical Specimens ◽

Human Dna ◽

Pathogen Dna ◽

Generation 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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Assessment of Metagenomic MinION and Illumina sequencing as an approach for the recovery of whole genome sequences of chikungunya and dengue viruses directly from clinical samples

10.1101/355560 ◽

2018 ◽

Cited By ~ 2

Author(s):

Liana E. Kafetzopoulou ◽

Kyriakos Efthymiadis ◽

Kuiama Lewandowski ◽

Ant Crook ◽

Dan Carter ◽

...

Keyword(s):

Dengue Virus ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Illumina Miseq ◽

Clinical Samples ◽

Whole Genome ◽

Metagenomic Sequencing ◽

Front Line ◽

Genome Sequences ◽

Oxford Nanopore

AbstractThe 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. We performed metagenomic sequencing using both the Illumina MiSeq and the portable Oxford Nanopore MinION to study the feasibility of whole genome sequencing from clinical samples containing chikungunya or dengue virus, two of the most important arboviruses. Direct metagenomic sequencing of nucleic acid extracts from serum and plasma without viral enrichment allowed for virus and coinfection identification, subtype determination and in the majority of cases elucidated complete or near-complete genomes adequate for phylogenetic analysis. This work demonstrates that metagenomic whole genome sequencing is feasible for over 90% and 80% of chikungunya and dengue virus PCR-positive patient samples respectively. It confirms the feasibility of field metagenomic sequencing for these and likely other RNA viruses, highlighting the applicability of this approach to front-line public health.

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A cell-free DNA metagenomic sequencing assay that integrates the damage response to infection

10.1101/648592 ◽

2019 ◽

Author(s):

Alexandre Pellan Cheng ◽

Philip Burnham ◽

John Richard Lee ◽

Matthew Pellan Cheng ◽

Manikkam Suthanthiran ◽

...

Keyword(s):

Urinary Tract ◽

High Throughput ◽

High Throughput Sequencing ◽

Viral Infections ◽

Clinical Samples ◽

Metagenomic Sequencing ◽

Kidney Transplant Recipients ◽

Bladder Tissue ◽

Cell Free Dna ◽

Free Dna

ABSTRACTHigh-throughput metagenomic sequencing offers an unbiased approach to identify pathogens in clinical samples. Conventional metagenomic sequencing however does not integrate information about the host, which is often critical to distinguish infection from infectious disease, and to assess the severity of disease. Here, we explore the utility of high-throughput sequencing of cell-free DNA after bisulfite conversion to map the tissue and cell types of origin of host-derived cell-free DNA, and to profile the bacterial and viral metagenome. We applied this assay to 51 urinary cfDNA isolates collected from a cohort of kidney transplant recipients with and without bacterial and viral infection of the urinary tract. We find that the cell and tissue types of origin of urinary cell-free DNA can be derived from its genome-wide profile of methylation marks, and strongly depend on infection status. We find evidence of kidney and bladder tissue damage due to viral and bacterial infection, respectively, and of the recruitment of neutrophils to the urinary tract during infection. Through direct comparison to conventional metagenomic sequencing as well as clinical tests of infection, we find this assay accurately captures the bacterial and viral composition of the sample. The assay presented here is straightforward to implement, offers a systems view into bacterial and viral infections of the urinary tract, and can find future use as a tool for the differential diagnosis of infections.

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Speed, accuracy, sensitivity and quality control choices for detecting clinically relevant microbes in whole blood from patients

10.1101/549477 ◽

2019 ◽

Author(s):

James Thornton ◽

George S. Watts ◽

Ken Youens-Clark ◽

Lee D. Cranmer ◽

Bonnie L. Hurwitz

Keyword(s):

Quality Control ◽

Febrile Neutropenia ◽

Relative Abundance ◽

Whole Blood ◽

Computer Time ◽

Health Concern ◽

Clinical Samples ◽

Metagenomic Sequencing ◽

Bacterial Mixtures ◽

Speed Accuracy

ABSTRACTInfections are a serious health concern worldwide, particularly in vulnerable populations such as the immunocompromised, elderly, and young. Advances in metagenomic sequencing availability, speed, and decreased cost offer the opportunity to supplement or replace culture-based identification of pathogens with DNA sequence-based diagnostics. Adopting metagenomic analysis for clinical use requires that all aspects of the pipeline are optimized and tested, including data analysis. We tested the accuracy, sensitivity, and resource requirements of Centrifuge within the context of clinically relevant bacteria. Binary mixtures of bacteria showed Centrifuge reliably identified organisms down to 0.1% relative abundance. A staggered mock bacterial community showed Centrifuge outperformed CLARK while requiring less computing resources. Shotgun metagenomes obtained from whole blood in three febrile neutropenia patients showed Centrifuge could identify both bacteria and viruses as part of a culture-free workflow. Finally, Centrifuge results changed minimally by eliminating time-consuming read quality control and host screening steps.AUTHOR SUMMARYImmunocompromised patients, such as those with febrile neutropenia (FN), are susceptible to infections, yet cultures fail to identify causative organisms ~80% of the time. High-throughput metagenomic sequencing offers a promising approach for identifying pathogens in clinical samples. Mining through metagenomes can be difficult given the volume of reads, overwhelming human contamination, and lack of well-defined bioinformatics methods. The goal of our study was to assess Centrifuge, a leading tool for the identification and quantitation of microbes, and provide a streamlined bioinformatics workflow real-word data from FN patient blood samples. To ensure the accuracy of the workflow we carefully examined each step using known bacterial mixtures that varied by genetic distance and abundance. We show that Centrifuge reliably identifies microbes present at just 1% relative abundance and requires substantially less computer time and resource than CLARK. Moreover, we found that Centrifuge results changed minimally by quality control and host-screening allowing for further reduction in compute time. Next, we leveraged Centrifuge to identify viruses and bacteria in blood draws for three FN patients, and confirmed suspected pathogens using genome coverage plots. We developed a web-based tool in iMicrobe and detailed protocols to promote re-use.

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