Analytical assessment of metagenomic workflows for pathogen detection with NIST RM 8376 and two sample matrices
ABSTRACT We assessed the analytical performance of metagenomic workflows using NIST Reference Material (RM) 8376 DNA from bacterial pathogens spiked into two simulated clinical samples: cerebrospinal fluid (CSF) and stool. Sequencing and taxonomic classification were used to generate signals for eac...
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American Society for Microbiology
2025-04-01
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| Series: | Microbiology Spectrum |
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| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.02806-24 |
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| author | Jason G. Kralj Stephanie L. Servetas Samuel P. Forry Monique E. Hunter Jennifer N. Dootz Scott A. Jackson |
| author_facet | Jason G. Kralj Stephanie L. Servetas Samuel P. Forry Monique E. Hunter Jennifer N. Dootz Scott A. Jackson |
| author_sort | Jason G. Kralj |
| collection | DOAJ |
| description | ABSTRACT We assessed the analytical performance of metagenomic workflows using NIST Reference Material (RM) 8376 DNA from bacterial pathogens spiked into two simulated clinical samples: cerebrospinal fluid (CSF) and stool. Sequencing and taxonomic classification were used to generate signals for each sample and taxa of interest and to estimate the limit of detection (LOD), the linearity of response, and linear dynamic range. We found that the LODs for taxa spiked into CSF ranged from approximately 100 to 300 copy/mL, with a linearity of 0.96 to 0.99. For stool, the LODs ranged from 10 to 221 kcopy/mL, with a linearity of 0.99 to 1.01. Furthermore, discriminating different E. coli strains proved to be workflow-dependent as only one classifier:database combination of the three tested showed the ability to differentiate the two pathogenic and commensal strains. Surprisingly, when we compared the linear response of the same taxa in the two different sample types, we found those functions to be the same, despite large differences in LODs. This suggests that the “agnostic diagnostic” theory for metagenomics (i.e., any organism can be identified because DNA is the measurand) may apply to different target organisms and different sample types. Because we are using RMs, we were able to generate quantitative analytical performance metrics for each workflow and sample set, enabling relatively rapid workflow screening before employing clinical samples. This makes these RMs a useful tool that will generate data needed to support the translation of metagenomics into regulated use.IMPORTANCEAssessing the analytical performance of metagenomic workflows, especially when developing clinical diagnostics, is foundational for ensuring that the measurements underlying a diagnosis are supported by rigorous characterization. To facilitate the translation of metagenomics into clinical practice, workflows must be tested using control samples designed to probe the analytical limitations (e.g., limit of detection). Spike-ins allow developers to generate fit-for-purpose control samples for initial workflow assessments and inform decisions about further development. However, clinical sample types include a wide range of compositions and concentrations, each presenting different detection challenges. In this work, we demonstrate how spike-ins elucidate workflow performance in two highly dissimilar sample types (stool and CSF), and we provide evidence that detection of individual organisms is unaffected by background sample composition, making detection sample-agnostic within a workflow. These demonstrations and performance insights will facilitate the translation of the technology to the clinic. |
| format | Article |
| id | doaj-art-e7ab083aaa624feabb7a0088ab64e67c |
| institution | DOAJ |
| issn | 2165-0497 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj-art-e7ab083aaa624feabb7a0088ab64e67c2025-08-20T03:06:53ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972025-04-0113410.1128/spectrum.02806-24Analytical assessment of metagenomic workflows for pathogen detection with NIST RM 8376 and two sample matricesJason G. Kralj0Stephanie L. Servetas1Samuel P. Forry2Monique E. Hunter3Jennifer N. Dootz4Scott A. Jackson5Complex Microbial Systems Group, Biosystems and Biomaterials Division, Materials Measurements Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland, USAComplex Microbial Systems Group, Biosystems and Biomaterials Division, Materials Measurements Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland, USAComplex Microbial Systems Group, Biosystems and Biomaterials Division, Materials Measurements Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland, USAComplex Microbial Systems Group, Biosystems and Biomaterials Division, Materials Measurements Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland, USAComplex Microbial Systems Group, Biosystems and Biomaterials Division, Materials Measurements Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland, USAComplex Microbial Systems Group, Biosystems and Biomaterials Division, Materials Measurements Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland, USAABSTRACT We assessed the analytical performance of metagenomic workflows using NIST Reference Material (RM) 8376 DNA from bacterial pathogens spiked into two simulated clinical samples: cerebrospinal fluid (CSF) and stool. Sequencing and taxonomic classification were used to generate signals for each sample and taxa of interest and to estimate the limit of detection (LOD), the linearity of response, and linear dynamic range. We found that the LODs for taxa spiked into CSF ranged from approximately 100 to 300 copy/mL, with a linearity of 0.96 to 0.99. For stool, the LODs ranged from 10 to 221 kcopy/mL, with a linearity of 0.99 to 1.01. Furthermore, discriminating different E. coli strains proved to be workflow-dependent as only one classifier:database combination of the three tested showed the ability to differentiate the two pathogenic and commensal strains. Surprisingly, when we compared the linear response of the same taxa in the two different sample types, we found those functions to be the same, despite large differences in LODs. This suggests that the “agnostic diagnostic” theory for metagenomics (i.e., any organism can be identified because DNA is the measurand) may apply to different target organisms and different sample types. Because we are using RMs, we were able to generate quantitative analytical performance metrics for each workflow and sample set, enabling relatively rapid workflow screening before employing clinical samples. This makes these RMs a useful tool that will generate data needed to support the translation of metagenomics into regulated use.IMPORTANCEAssessing the analytical performance of metagenomic workflows, especially when developing clinical diagnostics, is foundational for ensuring that the measurements underlying a diagnosis are supported by rigorous characterization. To facilitate the translation of metagenomics into clinical practice, workflows must be tested using control samples designed to probe the analytical limitations (e.g., limit of detection). Spike-ins allow developers to generate fit-for-purpose control samples for initial workflow assessments and inform decisions about further development. However, clinical sample types include a wide range of compositions and concentrations, each presenting different detection challenges. In this work, we demonstrate how spike-ins elucidate workflow performance in two highly dissimilar sample types (stool and CSF), and we provide evidence that detection of individual organisms is unaffected by background sample composition, making detection sample-agnostic within a workflow. These demonstrations and performance insights will facilitate the translation of the technology to the clinic.https://journals.asm.org/doi/10.1128/spectrum.02806-24metagenomicsreference materialspike-inpathogen detection |
| spellingShingle | Jason G. Kralj Stephanie L. Servetas Samuel P. Forry Monique E. Hunter Jennifer N. Dootz Scott A. Jackson Analytical assessment of metagenomic workflows for pathogen detection with NIST RM 8376 and two sample matrices Microbiology Spectrum metagenomics reference material spike-in pathogen detection |
| title | Analytical assessment of metagenomic workflows for pathogen detection with NIST RM 8376 and two sample matrices |
| title_full | Analytical assessment of metagenomic workflows for pathogen detection with NIST RM 8376 and two sample matrices |
| title_fullStr | Analytical assessment of metagenomic workflows for pathogen detection with NIST RM 8376 and two sample matrices |
| title_full_unstemmed | Analytical assessment of metagenomic workflows for pathogen detection with NIST RM 8376 and two sample matrices |
| title_short | Analytical assessment of metagenomic workflows for pathogen detection with NIST RM 8376 and two sample matrices |
| title_sort | analytical assessment of metagenomic workflows for pathogen detection with nist rm 8376 and two sample matrices |
| topic | metagenomics reference material spike-in pathogen detection |
| url | https://journals.asm.org/doi/10.1128/spectrum.02806-24 |
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