Clinical metagenomics

May combine two claims.

When spiked at reverse transcription, a panel of ZIKV-specific primers increased ZIKV reads 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 Mexico.

“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.”

• cites [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. sourceNot checked yet

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May combine two claims.

Almost all current conventional microbiological tests detect only one or a small panel of pathogens, or they require successful culture.

“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.”

Probably not a substantial claim: may not pass S4 (based).

mNGS is altering how clinicians diagnose and manage infectious diseases.

“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.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

mNGS is applied in areas such as drug resistance, microbial communities, host transcriptomics, and cancer.

“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.”

May combine two claims.

mNGS can drive precision diagnosis of infectious diseases and advance personalized patient care.

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

Classified as probably not a substantial claim.

Whether a microorganism found in a sample is a contaminant, colonizer, or genuine pathogen is frequently uncertain.

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

May combine two claims.

NGS data can be used for analyses beyond pathogen identification, including microbiome characterization and host transcriptome profiling.

“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).”

May combine two claims.

NGS assays are slower than conventional tests; a standard Illumina sequencing run alone takes more than 18 hours.

“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”

Probably not a substantial claim: may not pass S2 (checkable).

These applications are broad and potentially useful enough that diagnostic microbiology will probably be transformed in the near future.

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

• AssumptionRests on an assumption: Most mNGS applications have not yet entered routine clinical practice.

Classified as probably not a substantial claim. May combine two claims.

Metagenomic methods analyze all DNA or RNA in a sample, allowing examination of the whole microbiome and the human host genome or transcriptome.

“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.”

May combine two claims.

Cost, reimbursement, turnaround time, regulatory issues, and clinical utility remain major barriers to routine clinical mNGS in patient care.

“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.”

• cites [27] Greninger, A. The challenge of diagnostic metagenomics. Expert Rev. Mol. Diagn. 18, 605–615 (2018). sourceNot checked yet

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May combine two claims.

Clinical metagenomics applications include infectious disease diagnostics across syndromes and sample types, microbiome studies, host response transcriptomics, and detection of tumour-associated viruses and their integration sites.

“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).”

• cites Fig. 1Not checked yet

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• cites Table 1Not checked yet

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Probably not a substantial claim: may not pass S2 (checkable).

Detecting all potential pathogens and host responses in a sample has high potential utility for diagnosing infectious disease.

“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.”

Probably not a substantial claim: may not pass S4 (based).

Clinical metagenomic next-generation sequencing is moving rapidly from research into clinical laboratories.

“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.”

May combine two claims.

That case was the first demonstration that mNGS can provide clinically actionable information, because diagnosis led to targeted antibiotics and recovery.

“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.”

• 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. sourceNot checked yet

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May combine two claims.

Detecting nucleic acids by multiplex PCR or NGS does not prove that the identified microorganism causes the illness; results require clinical interpretation.

“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.”

May combine two claims.

Targeted NGS can improve sensitivity for the microorganisms targeted but narrows the range of pathogens that can be detected.

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

May combine two claims.

Targeted NGS methods increase how many pathogen reads appear in the data and their share of total reads.

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

May combine two claims.

mNGS was critical to the discovery of Merkel cell polyomavirus, now believed to cause Merkel cell carcinoma.

“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.”

• cites [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. sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

RNA-seq is commonly done on whole blood or PBMC samples, but any body fluid or tissue type may be suitable.

“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.”

May combine two claims.

Clinical mNGS usually focuses on microbial reads, but host gene expression analysis has a complementary role in studying infection.

“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).”

• cites [65] Kukurba, K. & Montgomery, S. RNA sequencing and analysis. Cold Spring Harb. Protoc. 2015, 951–969 (2015). sourceNot checked yet

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May combine two claims.

Compared with untargeted approaches, capture probe enrichment has drawbacks: bias toward targeted microorganisms, extra steps, higher costs, and long hybridization times of 24-48 hours.

“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.”

May combine two claims.

Machine-learning analyses of RNA-seq data have classified cancers, and translating these methods may help infectious diseases.

“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.”

• cites [76] Zhang, Y. et al. Identifying and analyzing different cancer subtypes using RNA-seq data of blood platelets. Oncotarget 8, 87494–87511 (2017). sourceNot checked yet

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May combine two claims.

That study provided high-resolution mapping of likely transmission events, some unexpected from initial epidemiology, and found putative resistance mutations in emerging resistant strains.

“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.”

• cites [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. sourceNot checked yet

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May combine two claims.

mNGS is a needle-in-a-haystack task: typically less than 1% of reads are non-human, and only some of those may be potential pathogens.

“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.”

May combine two claims.

In research, capture probe enrichment has improved sensitivity for detecting microbes, particularly viruses, but it is not yet used routinely in clinical diagnosis.

“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.”

May combine two claims.

Such genomes support subtyping and outbreak monitoring in hospitals for infection control or public health surveillance.

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

May combine two claims.

Several groups have validated mNGS in CLIA-certified clinical laboratories for diagnosing meningitis or encephalitis, sepsis, and pneumonia, and these assays are now available for clinical reference testing.

“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.”

• 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. sourceNot checked yet

  Not checked in the free run.

• cites [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. sourceNot checked yet

  Not checked in the free run.

• cites [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. sourceNot checked yet

  Not checked in the free run.

• 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). sourceNot checked yet

  Not checked in the free run.

• cites [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). sourceNot checked yet

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May combine two claims.

In oncology, NGS approaches that identify mutated genes can simultaneously detect cancer-associated viruses (herpesviruses, papillomaviruses, polyomaviruses) and/or gather virus-host interaction data.

“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.”

• cites [85] Nakagawa, H. & Fujita, M. Whole genome sequencing analysis for cancer genomics and precision medicine. Cancer Sci. 109, 513–522 (2018). sourceNot checked yet

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May combine two claims.

mNGS sensitivity depends critically on background level; tissues have more human host background than cell-free fluids, lowering microbial read counts and mNGS sensitivity.

“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.”

• cites [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. sourceNot checked yet

  Not checked in the free run.

• 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. sourceNot checked yet

  Not checked in the free run.

• cites [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. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Dysbiosis has been linked to obesity, diabetes mellitus, and inflammatory bowel disease, and altering the microbiome may treat these conditions.

“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.”

• cites [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). sourceNot checked yet

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May combine two claims.

This method has tracked Zika virus evolution and spread in the Americas and Ebola virus in West Africa, with some real-time monitoring affecting public health interventions.

“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.”

• cites [44] Faria, N. R. et al. Establishment and cryptic transmission of Zika virus in Brazil and the Americas. Nature 546, 406–410 (2017). sourceNot checked yet

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• cites [45] Grubaugh, N. et al. Genomic epidemiology reveals multiple introductions of Zika virus into the United States. Nature 546, 401–405 (2017). sourceNot checked yet

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• cites [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. sourceNot checked yet

  Not checked in the free run.

• cites [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. sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

The FDA has approved two NGS panels for clinical testing of actionable genomic aberrations in tumour samples.

“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.”

• cites [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). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Differential lysis methods can be limited by decreased sensitivity for microorganisms without cell walls, possible increase in exogenous contamination from additional reagents, and inability to detect free nucleic acid from dead organisms.

“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.”

• cites [101] Salter, S. et al. Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol. 12, 87 (2014). sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Microorganisms on this background list are either not reported or require higher reporting thresholds if clinically significant.

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

May combine two claims.

Human host background limitation may be overcome with brute force due to increasing sequencer capacities; e.g., astrovirus detected by ultradeep sequencing of brain tissue yielded 1,612 reads out of ~134 million (0.0012%).

“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.”

• cites [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). sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Clinical laboratory operations have defined workflows and scheduled staffing and are less amenable to on-demand testing than research laboratories.

“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.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Both differential lysis and propidium monoazide approaches would be cumbersome to implement in a highly reproducible fashion needed for clinical laboratory validation.

“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.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

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

“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.”

Probably not a substantial claim: may not pass S3 (load-bearing).

cfDNA analysis has been useful for non-invasive prenatal testing, such as identifying trisomy 21.

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

• cites [124] Norton, M. E. et al. Cell-free DNA analysis for noninvasive examination of trisomy. N. Engl. J. Med. 372, 1589–1597 (2015). sourceNot checked yet

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May combine two claims.

cfDNA analysis may have decreased sensitivity for predominantly intracellular pathogens (HTLV, Rickettsia spp., Pneumocystis jirovecii) and loses ability to interrogate cellular human host responses with RNA sequencing.

“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.”

May combine two claims.

ERCC RNA standards (or DNA equivalents) can be used as spike-in internal controls to control for assay inhibition and quantify pathogen titres by standard curve analysis.

“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.”

• cites [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). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S4 (based).

Laboratory surfaces, consumables and reagents are not DNA free.

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

May combine two claims.

Adding specific viral probes to NGS panels or sequencing whole tumour genome/exome would allow detection of integrated and exogenous viruses.

“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.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Unless fully automated sample-handling systems are available, wet lab manipulations for mNGS require considerable hands-on time and highly trained molecular biology staff.

“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.”

May combine two claims.

Implementing mNGS in clinical laboratory is complex and requires customizing research protocols with quality management consistent with regulatory standards.

“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.”

• cites [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. sourceNot checked yet

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Probably not a substantial claim: may not pass S2 (checkable).

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

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

Probably not a substantial claim: may not pass S2 (checkable), S4 (based).

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

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

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

There are ergonomic concerns with repetitive tasks like pipetting and potential for sample mix-up or omission of critical steps.

“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.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Internal spike-in control standards are available for other NGS applications, such as ERCC RNA standards composed of synthetic RNA oligonucleotides spanning a range of lengths and concentrations.

“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.”

• cites [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). sourceNot checked yet

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May combine two claims.

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 drugs, as evidenced by decreased risk of HCV-associated hepatocellular carcinoma after direct-acting antiviral treatment.

“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.”

• cites [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). sourceNot checked yet

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• cites [89] Kanwal, F. et al. Risk of hepatocellular cancer in HCV patients treated with direct-acting antiviral agents. Gastroenterology 153, 996–1005 (2017). sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

Because samples are typically handled in batches, batch analysis frequency is a major determinant of overall turnaround time.

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

May combine two claims.

FDA has general guidelines for clinical validation of NGS infectious disease testing, but no definitive recommendations or specific requirements for clinical implementation of mNGS.

“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.”

• cites [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 yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S4 (based).

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

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

May combine two claims.

Majority of library preparation methods for mNGS are performed manually and incur considerable staff time.

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

May combine two claims.

Establishing thresholds enables separation of true-positive matches from background, and thresholds can incorporate metrics such as read counts normalized to reads per million, external no-template controls or internal spike-in material, number of nonoverlapping genomic regions covered, and read abundance relative to negative controls.

“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”

Probably not a substantial claim: may not pass S3 (load-bearing).

Use of standardized reference materials and NGS data sets is helpful in comparative evaluation of different bioinformatics pipelines.

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

• cites [105] McIntyre, A. et al. Comprehensive benchmarking and ensemble approaches for metagenomic classifiers. Genome Biol. 18, 182 (2017). sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing).

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

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

• 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. sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

As NGS procedures become more standardized, there has been a drive towards increasing automation with liquid-handling biorobots.

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

• cites [115] May, M. Automated sample preparation. NIST Special Publication 1222, 1–17 (2016). sourceNot checked yet

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May combine two claims.

While current limitations suggest mNGS is unlikely to replace conventional diagnostics in short term, it can be a complementary, and perhaps essential, test in certain clinical situations.

“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.”

May combine two claims.

Overall cost is several hundreds to thousands of dollars per sample analysed, higher than many other clinical tests.

“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.”

May combine two claims.

Research studies have demonstrated clinical potential of nanopore sequencing in targeted universal 16S rRNA bacterial detection, microbiome analyses, whole-genome sequencing of bacteria and outbreak viruses, RNA-seq using standardized controls, and diagnosis of prosthetic joint and lower respiratory infections.

“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.”

• cites [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). sourceNot checked yet

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• cites [128] Kerkhof, L., Dillon, K., Häggblom, M. & McGuinness, L. Profiling bacterial communities by MinION sequencing of ribosomal operons. Microbiome 5, 116 (2017). sourceNot checked yet

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• cites [129] Tyler, A. et al. Evaluation of Oxford Nanopore’s MinION sequencing device for microbial whole genome sequencing applications. Sci. Rep. 8, 10931 (2018). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

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

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

• cites [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. sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Real-time sequencing 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 nanopore sequencing for Ebola and ZIKV surveillance and on ISS.

“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”

• cites [44] Faria, N. R. et al. Establishment and cryptic transmission of Zika virus in Brazil and the Americas. Nature 546, 406–410 (2017). sourceNot checked yet

  Not checked in the free run.

• cites [45] Grubaugh, N. et al. Genomic epidemiology reveals multiple introductions of Zika virus into the United States. Nature 546, 401–405 (2017). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

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

“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.”

• cites [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. sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Increased multiplexing is possible with greatly enhanced output from latest generation sequencers, such as Illumina NovaSeq.

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

May combine two claims.

ROC analysis is useful 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 set.

“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.”

• 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. sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Bioinformatics pipelines for mNGS analysis use many different algorithms, usually developed for research and constantly updated.

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

Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing), S4 (based). May combine two claims.

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 'non-infectious profile' can be treated for other causes.

“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.”

May combine two claims.

Larger and more complete databases like NCBI Nucleotide are more comprehensive but contain more errors than curated, more limited databases like FDA-ARGOS or RVDB.

“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.”

• cites [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 yet

  Not checked in the free run.

• cites [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). sourceNot checked yet

  Not checked in the free run.

• cites [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). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

A potential limitation with running larger numbers of samples per run is longer overall turnaround times for clinical use due to batch processing and workflow/computational considerations.

“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.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Many laboratories maintain both production and up-to-date development versions of clinical reference database, with production database updated at regular, prespecified intervals.

“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.”

Probably not a substantial claim: may not pass S2 (checkable).

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 from large patient cohorts.

“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.”

• cites [119] Afshinnekoo, E., Ahsanuddin, S. & Mason, C. E. Globalizing and crowdsourcing biomedical research. Br. Med. Bull. 120, 27–33 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [120] Brooks, J. P. et al. The truth about metagenomics: quantifying and counteracting bias in 16S rRNA studies. BMC Microbiol. 15, 66 (2015). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The selective sequencing method that depletes human reads during a run is not scalable at present, because the nanopore sequencer's throughput is limited (up to 10 million mNGS reads per MinION run as of 2019) and because reads must be matched to reference sequences computationally in real time.

“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”

• AssumptionRests on an assumption: The MinION nanopore sequencer can produce as many as ten million mNGS reads in a single run as of 2019.
• AssumptionRests on an assumption: The selective sequencing approach requires that reads be matched computationally to reference sequences in real time.

Probably not a substantial claim: may not pass S4 (based).

Untargeted approaches such as mNGS or whole-transcriptome RNA-seq may be limited by lower throughput of nanopore sequencing relative to short-read sequencing such as Illumina.

“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.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

To date, the only commercially available instruments for nanopore sequencing are from Oxford Nanopore Technologies and include MinION, GridION, PromethION.

“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).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Public databases for microbial reference genomes are continuously updated, and laboratories need to track exact versions used and deal with potential misannotations and other database errors.

“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.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

High-throughput processing of clinical samples for NGS may only be possible in reference laboratories.

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

Probably not a substantial claim: may not pass S3 (load-bearing).

A combined approach incorporating annotated sequences from multiple databases may enable greater confidence in sensitivity and specificity of microorganism identification.

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

May combine two claims.

It is usually necessary to make custom modifications to pipeline software and then lock down both software and reference databases for clinical validation.

“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.”

• cites [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. sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S4 (based).

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

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

May combine two claims.

Additional resources needed to run and maintain a bioinformatics analysis pipeline are considerable, and regulatory oversight steps can add notably to costs.

“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.”

Probably not a substantial claim: may not pass S3 (load-bearing).

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

“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.”

• cites [116] Levy, S. E. & Myers, R. M. Advancements in next-generation sequencing. Annu. Rev. Genomics Hum. Genet. 17, 95–115 (2016). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

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

“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”

• cites [27] Greninger, A. The challenge of diagnostic metagenomics. Expert Rev. Mol. Diagn. 18, 605–615 (2018). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Sensitivity for pathogen detection is decreased in clinical samples with high nucleic acid background or exceedingly low pathogen titres; this is only partially mitigated by increasing sequencing depth per sample as costs drop.

“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.”

May combine two claims.

Nanopore sequencing is an emerging NGS technology that enables real-time analysis of sequencing data.

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

• cites [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). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S4 (based).

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

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

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

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

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