B-148 Analytical Validation and Performance Characteristics of A Targeted Metagenomics Next-generation Sequencing Respiratory Pathogen ID/AMR Panel
Authors: Vest A , Houdeshell H , Arnold E , Harrison I , Hardison M , Clark C 
Published: September 2023

Background

The COVID-19 global pandemic underscored the importance of access to diagnostic testing, broad community surveillance, and genomic analysis of pathogen variants. The dearth of testing in the initial days and months of the pandemic highlighted the need to improve public health infrastructure. The subsequent onset of viral evolution, producing new and functionally different CoV2 strains, increased the importance for the availability and scalability of pathogen genome sequencing and data sharing pipelines through national surveillance programs. As SARS-CoV-2 moves from a pandemic to endemic state, the scientific and medical community must not forget the lessons learned and continue to reinforce the value of ongoing monitoring of circulating pathogens.

Methods

Aegis Sciences performed an analytical validation of the Respiratory Pathogen ID/AMR Target Enrichment Panel (RPIP, Illumina RUO), a next-generation sequencing assay that uses target capture-based enrichment for detection, quantification, and characterization of 282 viral, bacterial, and fungal respiratory pathogens and over 2000 antimicrobial resistance (AMR) genes. A total of 1598 validation samples were sequenced via the NextSeq 550 Dx (Illumina) and demultiplexed and assembled FASTQ files were analyzed with Explify RPIP Analysis application in BaseSpace Sequencing Hub (Illumina). Samples tested were contrived specimens using 60 different pathogen isolates various concentrations and clinical comparative samples, and the study included the evaluation of concentration vs genome coverage, inclusivity, exclusivity, and orthogonal analyses.

Results

RPIP is a highly sensitive, robust sequencing assay that can detect organisms at very low concentrations. Evaluation of copy number vs genome coverage revealed that 53 out of 60 organisms were detected at 1 copy/µL (PPV = 86.67%), and 60 out of 60 were detected at 10 copies/µL (PPV = 98.33%) and 50 copies/µL (PPV = 96.67%). The average coverage across the genome improved with increasing concentration and was 59.97% at 1 copy/µL, 72.45% at 10 copies/µL, and &gt;90% at 50 copies/µL. Inclusivity and exclusivity studies revealed an increase in false positives at low concentrations (1 copy/µL = 35.11%) and a reduction of false positives at higher concentrations (10 copies/µL = 20.3%) of isolates, and higher copy numbers of isolates allow for the true positives to be distinguished from contaminants and off-target organisms with very low coverage and quantification results. Orthogonal studies with upper respiratory clinical specimens tested previously by validated PCR methods revealed the following performance characteristics for respiratory pathogens, including SARS-CoV-2, influenza, parainfluenza, and rhinovirus: PPV = 81.11%, NPV = 99.66%, Sensitivity = 98.46%, and Specificity = 93.89%.

Conclusions

Completion of these studies demonstrated that next-generation sequencing is a cost effective, high-throughput option for broad pathogen tracking and surveillance. Laboratories, like Aegis, must continue to leverage collaborative relationships with public and private sector entities to expand resources, foster innovation, and broaden capabilities to quickly respond to biological threats.