Analytical Assessment of Metagenomic Workflows for Pathogen Detection with NIST RM® 8376 and Two Sample Matrices

Published

March 10, 2025

Author(s)

Jason Kralj, Stephanie Servetas, Samuel Forry, Monique Hunter, Jennifer Dootz, Scott Jackson

Abstract

We assessed the analytical performance of metagenomic workflows using NIST Reference Material® 8376 DNA from bacterial pathogens spiked into two simulated clinical samples: cerebral spinal fluid (CSF) and stool. Sequencing and taxonomic classification were used to generate signals for each sample and taxa of interest, and used to estimate the LOD, the response function, and linear dynamic range. We found that the LODs for taxa spiked into CSF ranged from approximately (0.1 to 0.3) copy/μL, with a linearity of 0.96 to 0.99. For stool, the LODs ranged from (10 to 221) copy/μL, with a linearity of 0.99 to 1.01. Further, discriminating different E. coli strains proved to be workflow-dependent, as only one classifier:database combination of the three tested showed the ability to differentate the two pathogenic and commensal strains. Surprisingly, when we compared the response functions 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 may apply to different target organisms and different sample types. 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 translation of metagenomics into regulated use.

Citation

Microbiology Spectrum

Volume

13

Issue

4

Pub Type

Journals

Download Paper

https://doi.org/10.1128/spectrum.02806-24

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Keywords

metagenomics, reference materials, microbiology, sequencing, pathogen detection

Citation

Kralj, J. , Servetas, S. , Forry, S. , Hunter, M. , Dootz, J. and Jackson, S. (2025), Analytical Assessment of Metagenomic Workflows for Pathogen Detection with NIST RM® 8376 and Two Sample Matrices, Microbiology Spectrum, [online], https://doi.org/10.1128/spectrum.02806-24, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=958249 (Accessed September 3, 2026)

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