Clinical metagenomics

In the paper

“this case was the first to demonstrate the utility of metagenomic NGS (mNGS) in providing clinically actionable information, as successful diagnosis prompted appropriate targeted antibiotic treatment and eventual recovery of the patient.”

What the sources say

• Partly supported

  The Case Report says the sequencing result prompted a switch to targeted penicillin G, and the Abstract reports discharge near the patient’s premorbid condition. The Discussion calls the diagnosis clinically actionable, but the paper does not establish that this was the first demonstration of mNGS providing such information (page numbers unavailable). (LLM, conf=0.87)

  cites [19] Wilson, M. R. et al. Actionable diagnosis of neuroleptospirosis by next-generation sequencing. N. Engl. J. Med. 370, 2408–2417 (2014). This case report describes the first use of clinical metagenomics for actionable diagnosis and treatment in a critically ill patient with a mysterious neurological infection. sourceExperimental — accuracy not yet measured

In the paper

“Nevertheless, several groups have successfully validated mNGS in Clinical Laboratory Improvement Amendments (CLIA)-certified clinical laboratories for the diagnosis of infections, including meningitis or encephalitis36,37, sepsis33,57 and pneumonia58, and these assays are now available for clinical reference testing of patients.”

What the sources say

• Supported

  The Abstract describes validation of an mNGS assay for meningitis and encephalitis in a licensed clinical laboratory. The Discussion says mNGS results are submitted to patients’ electronic medical records as interpretive reports, supporting clinical use. This citation is attached to meningitis or encephalitis; the sepsis and pneumonia examples have separate citations. (LLM, conf=0.86)

  cites [36] Miller, S. et al. Laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid. Preprint at bioRxiv https://doi.org/10.1101/330381 (2019). This paper describes the clinical validation of an mNGS assay for diagnosis of meningitis and encephalitis from cerebrospinal fluid. source
• Not checked yetcites [37] Schlaberg, R. et al. Validation of metagenomic next-generation sequencing tests for universal pathogen detection. Arch. Pathol. Lab Med. 141, 776–786 (2017). This paper summarizes the clinical validation of two mNGS assays for neurological infections and pneumonia. source
• Not checked yetcites [33] Blauwkamp, T. A. et al. Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease. Nat. Microbiol. https://doi.org/10.1038/s41564-018-0349-6 (2019). This paper describes the analytical and clinical validation of an mNGS assay for sepsis. source
• Partly supported

  The Results report that plasma NGS detected the fungus found in biopsy tissue in seven of nine patients with proven invasive fungal infection; the patient table includes one patient with septic shock. The paper does not establish that the assay was validated in a CLIA-certified laboratory for diagnosing sepsis or was available for clinical reference testing (Results and patient table; page unavailable). (LLM, conf=0.84)

  cites [57] Hong, D. et al. Liquid biopsy for infectious diseases: sequencing of cell-free plasma to detect pathogen DNA in patients with invasive fungal disease. Diagn. Microbiol. Infect. Dis. 92, 210–213 (2018). sourceExperimental — accuracy not yet measured
• Not checked yetcites [58] Schlaberg, R. et al. Viral pathogen detection by metagenomics and pan-viral group polymerase chain reaction in children with pneumonia lacking identifiable etiology. J. Infect. Dis. 215, 1407–1415 (2017). source

In the paper

“When spiked at the reverse transcription step, a panel of ZIKV-specific primers was found to increase the number of ZIKV reads by more than tenfold without appreciably decreasing broad metagenomic sensitivity for other pathogens, enabling whole-genome viral sequencing to characterize ZIKV spread from Brazil into Central America and Mexico46.”

What the sources say

• Not checked yetcites [46] Thézé, J. et al. Genomic epidemiology reconstructs the introduction and spread of Zika virus in central America and Mexico. Cell Host Microbe 23, 855–864 (2018). This study introduces the use of the metagenomic sequencing with spiked primer enrichment technique for simultaneous targeted and untargeted pathogen detection and genome assembly. source

In the paper

“However, nearly all conventional microbiological tests in current use detect only one or a limited panel of pathogens at a time or require that a microorganism be successfully cultured from a clinical sample.”

In the paper

“This emerging approach is changing how physicians diagnose and treat infectious disease, with applications spanning a wide range of areas, including antimicrobial resistance, the microbiome, human host gene expression (transcriptomics) and oncology.”

In the paper

“This emerging approach is changing how physicians diagnose and treat infectious disease, with applications spanning a wide range of areas, including antimicrobial resistance, the microbiome, human host gene expression (transcriptomics) and oncology.”

In the paper

“Thus, mNGS can be a key driver for precision diagnosis of infectious diseases, advancing precision medicine efforts to personalize patient care in this field.”

In the paper

“it is often unclear whether a detected microorganism is a contaminant, colonizer or bona fide pathogen.”

In the paper

“Another key advantage of NGS approaches is that the sequencing data can potentially be leveraged for additional analyses beyond the mere identification of a causative pathogen, such as microbiome characterization and parallel analyses of human host responses through transcriptome profiling by RNA sequencing (RNA-seq).”

In the paper

“while NGS assays in current use cannot compare with conventional tests with respect to speed — the sequencing run alone on a standard Illumina instrument takes >18 hours”

In the paper

“Nonetheless, the breadth and potential clinical utility of these applications are likely to transform the field of diagnostic microbiology in the near future.”

In the paper

“While most molecular assays target only a limited number of pathogens using specific primers or probes, metagenomic approaches characterize all DNA or RNA present in a sample, enabling analysis of the entire microbiome as well as the human host genome or transcriptome in patient samples.”

In the paper

“Considerations of cost, reimbursement, turnaround time, regulatory considerations and, perhaps most importantly, clinical utility also remain major hurdles for the routine implementation of clinical mNGS in patient care settings27.”

What the sources say

• Not checked yetcites [27] Greninger, A. The challenge of diagnostic metagenomics. Expert Rev. Mol. Diagn. 18, 605–615 (2018). source

In the paper

“To date, applications of clinical metagenomics have included infectious disease diagnostics for a variety of syndromes and sample types, microbiome analyses in both diseased and healthy states, characterization of the human host response to infection by transcriptomics and the identification of tumour-associated viruses and their genomic integration sites (Fig. 1; Table 1).”

What the sources say

• Not checked yetcites Fig. 1
• Not checked yetcites Table 1

In the paper

“The capacity to detect all potential pathogens — bacteria, viruses, fungi and parasites — in a sample and simultaneously interrogate host responses has great potential utility in the diagnosis of infectious disease.”

In the paper

“Clinical metagenomic next-generation sequencing (mNGS), the comprehensive analysis of microbial and host genetic material (DNA and RNA) in samples from patients, is rapidly moving from research to clinical laboratories.”

In the paper

“Of course, detection of nucleic acids, either by multiplex PCR panels or NGS, does not by itself prove that an identified microorganism is the cause of the illness, and findings have to be interpreted in the clinical context.”

In the paper

“This step can increase the detection sensitivity for microorganisms being targeted, although it limits the breadth of potential pathogens that can be identified.”

In the paper

“Targeted approaches have the benefit of increasing the number and proportion of pathogen reads in the sequence data.”

In the paper

“For example, mNGS was critical in the discovery of Merkel cell polyomavirus (Fig. 1d), now believed to be the cause of Merkel cell carcinoma, a rare skin cancer seen most commonly in elderly patients86.”

What the sources say

• Not checked yetcites [86] Feng, H., Shuda, M., Chang, Y. & Moore, P. Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science 319, 1096–1100 (2008). This paper describes the discovery of a novel polyomavirus associated with a rare skin cancer using NGS. source

In the paper

“Although RNA-seq analyses are commonly performed on whole blood or peripheral blood mononuclear cell (PBMC) samples, any body fluid or tissue type is potentially amenable to these analyses.”

In the paper

“Clinical mNGS typically focuses on microbial reads; however, there is a complementary role for the analysis of gene expression in studying human host responses to infection65 (Fig. 1c).”

What the sources say

• Not checked yetcites [65] Kukurba, K. & Montgomery, S. RNA sequencing and analysis. Cold Spring Harb. Protoc. 2015, 951–969 (2015). source

In the paper

“However, drawbacks of capture probe enrichment, compared with untargeted approaches for infectious disease diagnosis, include a bias towards targeted microorganisms, added steps, increased costs and long hybridization times (24–48 hours) as a result of the additional processing needed for maximal efficiency.”

In the paper

“Machine-learning-based analyses of RNA-seq data have been used for cancer classification76, and translation of these approaches may be promising for infectious diseases.”

What the sources say

• Not checked yetcites [76] Zhang, Y. et al. Identifying and analyzing different cancer subtypes using RNA-seq data of blood platelets. Oncotarget 8, 87494–87511 (2017). source

In the paper

“This study demonstrated for the first time the high-resolution mapping of likely transmission events in a hospital, some of which were unexpected on the basis of initial epidemiological data, and also identified putative resistance mutations in emerging resistant strains.”

What the sources say

• Not checked yetcites [55] Snitkin, E. et al. Tracking a hospital outbreak of carbapenem-resistant Klebsiella pneumoniae with whole-genome sequencing. Sci. Transl Med. 4, 148ra116 (2012). This study is the first to demonstrate the potential of whole-genome bacterial sequencing using NGS to track transmission of a hospital outbreak of carbapenem-resistant K. pneumoniae. source

In the paper

“mNGS is a needle-in-a-haystack endeavour, as only a small proportion (typically <1%) of reads are non-human, of which only a subset may correspond to potential pathogens.”

In the paper

“Although this enrichment method has been shown to increase the sensitivity of metagenomic detection in research settings, especially for viruses, it has yet to be used routinely for clinical diagnosis.”

In the paper

“These genome sequences are then used for subtyping and/or monitoring hospital outbreaks in support of infection control and/or public health surveillance efforts.”

In the paper

“In oncology, whole-genome or directed NGS approaches to identify mutated genes can be used to simultaneously uncover viruses associated with cancer (that is, herpesviruses, papillomaviruses and polyomaviruses) and/or to gather data on virus–host interactions85.”

What the sources say

• Not checked yetcites [85] Nakagawa, H. & Fujita, M. Whole genome sequencing analysis for cancer genomics and precision medicine. Cancer Sci. 109, 513–522 (2018). source

In the paper

“A limitation of mNGS is that the sensitivity of the approach is critically dependent on the level of background. Tissues, for example, have increased human host background relative to cell-free body fluids, resulting in a reduced number and proportion of microbial reads and hence a decrease in mNGS sensitivity33,36,37.”

What the sources say

• Not checked yetcites [33] Blauwkamp, T. A. et al. Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease. Nat. Microbiol. https://doi.org/10.1038/s41564-018-0349-6 (2019). This paper describes the analytical and clinical validation of an mNGS assay for sepsis. source
• Not checked yetcites [36] Miller, S. et al. Laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid. Preprint at bioRxiv https://doi.org/10.1101/330381 (2019). This paper describes the clinical validation of an mNGS assay for diagnosis of meningitis and encephalitis from cerebrospinal fluid. source
• Not checked yetcites [37] Schlaberg, R. et al. Validation of metagenomic next-generation sequencing tests for universal pathogen detection. Arch. Pathol. Lab Med. 141, 776–786 (2017). This paper summarizes the clinical validation of two mNGS assays for neurological infections and pneumonia. source

In the paper

“Alterations of the microbiome, known as dysbiosis, have also been shown to be related to obesity, diabetes mellitus and inflammatory bowel disease64, and manipulation of the microbiome may be a pathway to treating these pathological conditions.”

What the sources say

• Not checked yetcites [64] Boulangé, C., Neves, A., Chilloux, J., Nicholson, J. & Dumas, M. Impact of the gut microbiota on inflammation, obesity, and metabolic disease. Genome Med. 8, 42 (2016). source

In the paper

“This method has been used to track the evolution and spread of Zika virus (ZIKV) in the Americas44–46 and of Ebola virus in West Africa47, with some demonstrations of real-time monitoring having an impact on public health interventions.”

What the sources say

• Not checked yetcites [44] Faria, N. R. et al. Establishment and cryptic transmission of Zika virus in Brazil and the Americas. Nature 546, 406–410 (2017). source
• Not checked yetcites [45] Grubaugh, N. et al. Genomic epidemiology reveals multiple introductions of Zika virus into the United States. Nature 546, 401–405 (2017). source
• Not checked yetcites [46] Thézé, J. et al. Genomic epidemiology reconstructs the introduction and spread of Zika virus in central America and Mexico. Cell Host Microbe 23, 855–864 (2018). This study introduces the use of the metagenomic sequencing with spiked primer enrichment technique for simultaneous targeted and untargeted pathogen detection and genome assembly. source
• Not checked yetcites [47] Quick, J. et al. Real-time, portable genome sequencing for Ebola surveillance. Nature 530, 228–232 (2016). This study describes deployment of a portable nanopore sequencer for real-time actionable sequencing of clinical samples during the Ebola outbreak in West Africa. source

In the paper

“To date, the US Food and Drug Administration (FDA) has approved the clinical use of two NGS panels testing for actionable genomic aberrations in tumour samples87.”

What the sources say

• Not checked yetcites [87] Allegretti, M. et al. Tearing down the walls: FDA approves next generation sequencing (NGS) assays for actionable cancer genomic aberrations. J. Exp. Clin. Cancer Res. 37, 47 (2018). source

In the paper

“These limitations include potential decreased sensitivity for microorganisms without cell walls, such as Mycoplasma spp. or parasites; a possible paradoxical increase in exogenous background contamination by use of additional reagents101; and the inability to detect free nucleic acid from dead organisms that are lysed in vivo by human host immune cells or antibiotic treatment.”

What the sources say

• Not checked yetcites [101] Salter, S. et al. Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol. 12, 87 (2014). source

In the paper

“Microorganisms on this list are either not reported or will require higher thresholds for reporting if they are clinically significant organisms.”

In the paper

“For instance, an astrovirus was detected in a child with encephalitis by ultradeep sequencing of brain tissue, yielding only 1,612 reads out of ~134 million (0.0012%) sequences103.”

What the sources say

• Not checked yetcites [103] Naccache, S. et al. Diagnosis of neuroinvasive astrovirus infection in an immunocompromised adult with encephalitis by unbiased next-generation sequencing. Clin. Infect. Dis. 60, 919–923 (2015). source

In the paper

“Clinical laboratory operations are characterized by a defined workflow with scheduled staffing levels and are less amenable to on-demand testing than those of research laboratories.”

In the paper

“In general, both the differential lysis and propidium monoazide approaches would also be cumbersome to implement in a highly reproducible fashion, which is needed for clinical laboratory validation.”

In the paper

“Maintaining high quality during complex mNGS procedures can be stressful to staff, as slight deviations in sample handling can lead to major changes in the results generated.”

In the paper

“Similarly, cfDNA analysis has been useful for non-invasive prenatal testing applications, such as for the identification of trisomy 21 (ref.124).”

What the sources say

• Not checked yetcites [124] Norton, M. E. et al. Cell-free DNA analysis for noninvasive examination of trisomy. N. Engl. J. Med. 372, 1589–1597 (2015). source

In the paper

“However, limitations of cfDNA analysis may include decreased sensitivity for detection of predominantly intracellular pathogens, such as human T cell lymphotropic virus, Rickettsia spp. and Pneumocystis jirovecii, and loss of the ability to interrogate cellular human host responses with RNA sequencing.”

In the paper

“The complete set or a portion of the ERCC RNA standards (or their DNA equivalents) can be used as spike-in internal controls to control for assay inhibition and to quantify titres of detected pathogens by standard curve analysis108.”

What the sources say

• Not checked yetcites [108] Avraham, R. et al. A highly multiplexed and sensitive RNA-seq protocol for simultaneous analysis of host and pathogen transcriptomes. Nat. Protoc. 11, 1477–1491 (2016). source

In the paper

“In addition, laboratory surfaces, consumables and reagents are not DNA free.”

In the paper

“Detection of reads corresponding to both integrated and exogenous viruses in these samples would be possible with the addition of specific viral probes to the panel or accomplished incidentally while sequencing the whole tumour genome or exome.”

In the paper

“Unless fully automated sample-handling systems are readily available, wet lab manipulations for mNGS require considerable hands-on time to perform, as well as clinical staff who are highly trained in molecular biology procedures.”

In the paper

“Implementation of mNGS in the clinical laboratory is a complex endeavour that requires customization of research protocols using a quality management approach consistent with regulatory standards90.”

What the sources say

• Not checked yetcites [90] Burd, E. Validation of laboratory-developed molecular assays for infectious diseases. Clin. Microbiol. Rev. 23, 550–576 (2010). This paper summarizes the essential requirements for validation of infectious disease assays in a clinical laboratory. source

In the paper

“There is a critical need for standardized reference organisms and genomic materials to facilitate such comparisons and to define optimal analysis methods.”

In the paper

“Well-characterized reference standards and controls are needed to ensure mNGS assay quality and stability over time.”

In the paper

“There are ergonomic concerns with repetitive tasks such as pipetting, as well as potential for inadvertent sample mix-up or omission of critical steps in the workflow.”

In the paper

“Internal spike-in control standards are available for other NGS applications such as transcriptome analysis by RNA-seq, with External RNA Controls Consortium (ERCC) RNA standards composed of synthetic RNA oligonucleotides spanning a range of nucleotide lengths and concentrations107.”

What the sources say

• Not checked yetcites [107] Pine, P. et al. Evaluation of the External RNA Controls Consortium (ERCC) reference material using a modified Latin square design. BMC Biotechnol. 16, 54 (2016). source

In the paper

“Additional knowledge of integrated or active viral infections in cancers and their involvement in signalling pathways may inform preventive and therapeutic interventions with targeted antiviral and/or chemotherapeutic drugs88, as evidenced by the decreased risk of hepatitis C virus-associated hepatocellular carcinoma after treatment with direct-acting antiviral agents89.”

What the sources say

• Not checked yetcites [88] Saha, A., Kaul, R., Murakami, M. & Robertson, E. S. Tumor viruses and cancer biology: modulating signaling pathways for therapeutic intervention. Cancer Biol. Ther. 10, 961–978 (2010). source
• Not checked yetcites [89] Kanwal, F. et al. Risk of hepatocellular cancer in HCV patients treated with direct-acting antiviral agents. Gastroenterology 153, 996–1005 (2017). source

In the paper

“As samples are typically handled in batches, the frequency of batch analysis is a major determinant of overall turnaround time.”

In the paper

“Although the FDA has provided general guidelines for clinical validation of NGS infectious disease testing91, there are no definitive recommendations for the clinical implementation of mNGS testing, nor is there mention of specific requirements.”

What the sources say

• Not checked yetcites [91] Food and Drug Administration. Infectious disease next generation sequencing based diagnostic devices: microbial identification and detection of antimicrobial resistance and virulence markers (FDA, 2016). This draft guidance from the FDA covers considerations for validation and approval of sequencing-based diagnostic devices for infectious diseases.

In the paper

“User-friendly bioinformatics software for analysis of mNGS data is not currently available.”

In the paper

“Thus, the majority of library preparation methods for mNGS are performed manually and hence incur considerable staff time.”

In the paper

“Establishing thresholds enables separation of true-positive matches from the background, and these thresholds can incorporate metrics such as the number of sequence reads aligning to the detected microorganism, normalized to reads per million, external no-template control samples or internal spike-in material; the number of nonoverlapping genomic regions covered; and the read abundance in clinical”

In the paper

“The use of standardized reference materials and NGS data sets is also helpful in comparative evaluation of different bioinformatics pipelines105.”

What the sources say

• Not checked yetcites [105] McIntyre, A. et al. Comprehensive benchmarking and ensemble approaches for metagenomic classifiers. Genome Biol. 18, 182 (2017). source

In the paper

“Ongoing monitoring is particularly important for mNGS assays to verify acceptable performance over time and to investigate atypical findings36.”

What the sources say

• Not checked yetcites [36] Miller, S. et al. Laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid. Preprint at bioRxiv https://doi.org/10.1101/330381 (2019). This paper describes the clinical validation of an mNGS assay for diagnosis of meningitis and encephalitis from cerebrospinal fluid. source

In the paper

“As NGS procedures become more standardized, there has been a drive towards increasing automation with the use of liquid-handling biorobots115.”

What the sources say

• Not checked yetcites [115] May, M. Automated sample preparation. NIST Special Publication 1222, 1–17 (2016). source

In the paper

“In summary, while current limitations suggest that mNGS is unlikely to replace conventional diagnostics in the short term, it can be a complementary, and perhaps essential, test in certain clinical situations.”

In the paper

“This leads to an overall cost of several hundreds to thousands of dollars per sample analysed, which is higher than that for many other clinical tests.”

In the paper

“Research studies have also demonstrated the clinical potential of nanopore sequencing in targeted universal 16S ribosomal RNA (rRNA) bacterial detection127, microbiome analyses128, whole-genome sequencing of bacteria129 and outbreak viruses44,45,47, RNA sequencing (RNA-seq) using standardized controls130 and diagnosis of prosthetic joint131 and lower respiratory infections99.”

What the sources say

• Not checked yetcites [127] Mitsuhashi, S. et al. A portable system for rapid bacterial composition analysis using a nanopore-based sequencer and laptop computer. Sci. Rep. 7, 5657 (2017). source
• Not checked yetcites [128] Kerkhof, L., Dillon, K., Häggblom, M. & McGuinness, L. Profiling bacterial communities by MinION sequencing of ribosomal operons. Microbiome 5, 116 (2017). source
• Not checked yetcites [129] Tyler, A. et al. Evaluation of Oxford Nanopore’s MinION sequencing device for microbial whole genome sequencing applications. Sci. Rep. 8, 10931 (2018). source

In the paper

“Clinical laboratories are highly regulated, and general laboratory and testing requirements apply to all molecular diagnostic assays reported for patient care90.”

What the sources say

• Not checked yetcites [90] Burd, E. Validation of laboratory-developed molecular assays for infectious diseases. Clin. Microbiol. Rev. 23, 550–576 (2010). This paper summarizes the essential requirements for validation of infectious disease assays in a clinical laboratory. source

In the paper

“Real-time sequencing in particular may be a game-changing technology for point-of-care applications in clinical medicine and public health, as laboratories have begun to apply these tools to diagnose atypical infections and track pathogen outbreaks, as demonstrated by the recent deployment of real-time nanopore sequencing for remote epidemiological surveillance of Ebola44 and ZIKV44,45, and even”

What the sources say

• Not checked yetcites [44] Faria, N. R. et al. Establishment and cryptic transmission of Zika virus in Brazil and the Americas. Nature 546, 406–410 (2017). source
• Not checked yetcites [45] Grubaugh, N. et al. Genomic epidemiology reveals multiple introductions of Zika virus into the United States. Nature 546, 401–405 (2017). source

In the paper

“Although the use of mNGS for informing clinical care has been demonstrated in multiple case reports and small case series118, nearly all studies have been retrospective, and clinical utility has yet to be established in a large-scale prospective clinical trial.”

What the sources say

• Not checked yetcites [118] Simner, P. J., Miller, S. & Carroll, K. C. Understanding the promises and hurdles of metagenomic next-generation sequencing as a diagnostic tool for infectious diseases. Clin. Infect. Dis. 66, 778–788 (2018). This is a concise yet comprehensive review of some of the clinical applications of mNGS for diagnosis of infectious diseases. source

In the paper

“Increased multiplexing is also possible with the greatly enhanced output from the latest generation of sequencers, such as the Illumina NovaSeq instruments.”

In the paper

“Receiver–operator curve (ROC) analysis is a useful tool to determine optimal threshold values for a training set of clinical samples with known results, with verification of pre-established thresholds using an independent validation set36.”

What the sources say

• Not checked yetcites [36] Miller, S. et al. Laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid. Preprint at bioRxiv https://doi.org/10.1101/330381 (2019). This paper describes the clinical validation of an mNGS assay for diagnosis of meningitis and encephalitis from cerebrospinal fluid. source

In the paper

“Bioinformatics pipelines for mNGS analysis use a number of different algorithms, usually developed for the research setting and constantly updated by software developers.”

In the paper

“For example, the mNGS transcriptomic approach might enable effective treatment triage, whereby antimicrobials are only needed for patients showing an ‘infectious profile’ of gene expression and those with a ‘non-infectious profile’ can be treated for other causes.”

In the paper

“Larger and more complete databases containing publicly deposited sequences such as the National Center for Biotechnology Information (NCBI) Nucleotide database are more comprehensive but also contain more errors than curated, more limited databases such as FDA-ARGOS91,113 or the FDA Reference Viral Database (RVDB)114.”

What the sources say

• Not checked yetcites [91] Food and Drug Administration. Infectious disease next generation sequencing based diagnostic devices: microbial identification and detection of antimicrobial resistance and virulence markers (FDA, 2016). This draft guidance from the FDA covers considerations for validation and approval of sequencing-based diagnostic devices for infectious diseases.
• Not checked yetcites [113] Goldberg, B., Sichtig, H., Geyer, C., Ledeboer, N. & Weinstock, G. Making the leap from research laboratory to clinic: challenges and opportunities for next-generation sequencing in infectious disease diagnostics. mBio 6, e01888-15 (2015). source
• Not checked yetcites [114] Goodacre, N., Aljanahi, A., Nandakumar, S., Mikailov, M. & Khan, A. S. A reference viral database (RVDB) to enhance bioinformatics analysis of high-throughput sequencing for novel virus detection. mSphere 3, e00069-18 (2018). source

In the paper

“However, a potential limitation with running larger numbers of samples per run is longer overall turnaround times for clinical use owing to the requirement for batch processing as well as sample workflow and computational analysis considerations.”

In the paper

“Many laboratories maintain both production and up-to-date development versions of the clinical reference database (for example, the NCBI nucleotide database is updated every 2 weeks), with the production database being updated at regular, prespecified intervals.”

In the paper

“High-quality evidence that clinical metagenomic assays are effective in guiding patient management will require protocols that minimize potential assay and patient selection bias and compare relevant health outcomes using data sets generated from large patient cohorts119,120.”

What the sources say

• Not checked yetcites [119] Afshinnekoo, E., Ahsanuddin, S. & Mason, C. E. Globalizing and crowdsourcing biomedical research. Br. Med. Bull. 120, 27–33 (2016). source
• Not checked yetcites [120] Brooks, J. P. et al. The truth about metagenomics: quantifying and counteracting bias in 16S rRNA studies. BMC Microbiol. 15, 66 (2015). source

In the paper

“Although attractive for purposes of protecting patient privacy and confidentiality, as human reads are depleted as part of the sequencing run, this approach is not currently scalable owing to the limited throughput of the nanopore sequencer to date (up to 10 million mNGS reads per run on the MinION nanopore sequencer as of 2019) and the need to computationally match reads to reference sequences in”

In the paper

“Untargeted approaches such as mNGS or whole-transcriptome RNA-seq, however, may be limited by the lower throughput of nanopore sequencing relative to short-read sequencing such as with an Illumina instrument.”

In the paper

“To date, the only commercially available instruments for nanopore sequencing are from Oxford Nanopore Technologies and include the MinION (1 flow cell), GridION (5 flow cell capacity) and PromethION (48 flow cell capacity).”

In the paper

“Additionally, public databases for microbial reference genomes are being continuously updated, and laboratories need to keep track of the exact versions used in addition to dealing with potential misannotations and other database errors.”

In the paper

“Additionally, high-throughput processing of clinical samples for NGS may only be possible in reference laboratories.”

In the paper

“A combined approach that incorporates annotated sequences from multiple databases may enable greater confidence in the sensitivity and specificity of microorganism identification.”

In the paper

“As for wet lab procedures, it is usually necessary to make custom modifications to the pipeline software and then lock down both the software and reference databases for the purposes of clinical validation112.”

What the sources say

• Not checked yetcites [112] Roy, S. et al. Standards and guidelines for validating next-generation sequencing bioinformatics pipelines: a joint recommendation of the Association for Molecular Pathology and the College of American Pathologists. J. Mol. Diagn. 20, 4–27 (2018). This draft guidance from the Association for Molecular Pathology and College of American Pathologists reviews standards and guidelines for validation of NGS bioinformatics pipelines. source

In the paper

“Technological advancements in library preparation methods, sequence generation and computational bioinformatics are enabling quicker and more comprehensive metagenomic analyses at lower cost.”

In the paper

“The additional resources needed to run and maintain a bioinformatics analysis pipeline are also considerable, and steps taken to ensure regulatory oversight can add notably to costs as well.”

In the paper

“The development of microfluidic devices for NGS sample library preparation, such as VolTRAX116, could eventually enable clinicians to use mNGS more widely in hospital laboratories or point-of-care settings.”

What the sources say

• Not checked yetcites [116] Levy, S. E. & Myers, R. M. Advancements in next-generation sequencing. Annu. Rev. Genomics Hum. Genet. 17, 95–115 (2016). source

In the paper

“As a comprehensive direct detection method, mNGS may eventually replace culture, antigen detection and PCR methods in clinical microbiology, but indirect approaches such as viral serological testing will continue to play a key part in the diagnostic work-up for infections27, and functional assays such as culture and phenotypic susceptibility testing will likely always be useful for research”

What the sources say

• Not checked yetcites [27] Greninger, A. The challenge of diagnostic metagenomics. Expert Rev. Mol. Diagn. 18, 605–615 (2018). source

In the paper

“In particular, sensitivity for pathogen detection is decreased in clinical samples with a high nucleic acid background or with exceedingly low pathogen titres; this concern is only partially mitigated by increasing sequencing depth per sample as costs continue to drop.”

In the paper

“Nanopore sequencing is an emerging next-generation sequencing (NGS) technology that enables real-time analysis of sequencing data125.”

What the sources say

• Not checked yetcites [125] Jain, M., Olsen, H., Paten, B. & Akeson, M. The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biol. 17, 239 (2016). source

In the paper

“Most laboratories lack the robotic equipment and established automated protocols to multiplex large numbers of patient samples in a single run.”

In the paper

“Typically, two biorobots are needed for clinical mNGS for both the pre-amplification and post-amplification steps to avoid PCR amplicon cross-contamination.”