UMPlex™: a targeted next-generation sequencing primer design workflow

TL;DR

UMPlex™ is a new primer design workflow for targeted next-generation sequencing that improves pathogen detection in clinical diagnostics.

Contribution

A systematic primer validation methodology that enhances tNGS performance by addressing amplification inconsistencies.

Findings

tNGS outperformed TaqMan Array in detecting pathogens in respiratory samples with influenza-like symptoms.

tNGS showed high specificity and efficacy with 11 cultured pathogens isolated exclusively.

tNGS produced consistent results with metagenomic NGS while generating less data.

Abstract

We have developed a tailored next-generation sequencing (tNGS) panel, employing our innovative UMPlex™ primer design workflow, to enhance pathogen identification in clinical diagnostics. Through iterative experimentation and rigorous validation, we refined the primer design by excluding those with insufficient specificity or efficiency. To mitigate amplification challenges arising from pathogenic mutations, we implemented a strategy of using a minimum of two primer pairs per pathogen, ensuring redundancy and robust detection. Validation using clinical samples showcased high specificity and efficacy, with 11 cultured pathogens isolated exclusively. In a study involving 107 positive respiratory samples, tNGS outperformed the TaqMan Array, detecting a higher number of pathogens in patients with influenza-like symptoms of unknown etiology. Additionally, tNGS yielded higher read counts for potentially pathogenic microorganisms and produced results consistent with metagenomic NGS, despite generating a reduced data volume. This approach not only improves detection rates but also offers a flexible tool for both clinical diagnostics and surveillance, particularly in the context of influenza-like illnesses.

The online version contains supplementary material available at 10.1186/s12985-025-02831-6.

Rapid and accurate pathogen identification is essential for effective diagnosis and treatment of infectious diseases.

Targeted next-generation sequencing (tNGS) offers a revolutionary approach for comprehensive pathogen detection.

Our pioneering work introduces a systematic primer validation methodology, addressing amplification inconsistencies in tNGS designs.

This method has been successfully validated in influenza-like patients, demonstrating its efficacy and reliability.

The online version contains supplementary material available at 10.1186/s12985-025-02831-6.

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