This case study demonstrates the performance of Meridian’s molecular solutions within a customer’s amplicon-based targeted NGS assay covering 177 BRCA regions associated with breast cancer. The developer, already using Meridian’s mix for qPCR workflows, aimed to evaluate whether the same chemistry could be applied directly to tNGS without modification.
APPLICATION NOTE qPCR MIXES FOR tNGS APPLICATION NOTE
Inside an Assay Developer’s Initial Study Comparing qPCR Mixes for Targeted NGS INTRODUCTION Next-generation sequencing (NGS) has transformed genomic research and clinical diagnostics by enabling high-throughput, comprehensive genetic analysis, paving the way for precision medicine. However, traditional whole-genome sequencing (WGS) and whole-exome sequencing (WES) approaches can be costly, time-intensive and require large amounts of input DNA—limitations that are particularly challenging for clinical applications such as cancer screening, liquid biopsy and hereditary disease diagnostics. Targeted NGS (tNGS) provides a practical alternative to WGS by concentrating on specific genes or regions, enabling deep coverage for known genetic mutations. This focused approach delivers high variant detection sensitivity across large panels while reducing overall sequencing costs. 1 H owever, to fully realize these benefits, tNGS workflows must incorporate optimized amplification strategies—particularly for multiplex PCR-based methods. Without careful optimization, challenges such as primer-dimer formation, GC-content variability, and inefficient polymerase performance can lead
to uneven coverage and reduced accuracy. 2 Overcoming these hurdles requires robust enzyme chemistry designed to deliver consistent, reliable results at every step. This study demonstrates the performance of Meridian’s molecular solutions within a customer’s amplicon-based targeted NGS assay covering 177 BRCA regions associated with breast cancer. The developer, already using Meridian’s mix for qPCR workflows, aimed to evaluate whether the same chemistry could be applied directly to tNGS without modification. To test this, they substituted Meridian’s Lyo- Ready Direct DNA qPCR Blood Mix [MDX122] into their established 177-amplicon BRCA panel and benchmarked its performance against a commercial reference mix.
ABOUT MERIDIAN
Meridian is a global provider of innovative diagnostic solutions and critical raw materials for molecular and immunoassay development. We design, manufacture and deliver components that enable fast, accurate and reliable diagnostics—from early research through commercial testing. As part of our molecular focus, we offer a comprehensive portfolio of: • Enzymes for isothermal, qPCR and NGS sample prep
Our solutions, engineered for workflow efficiency, sensitivity and reproducibility, enable applications from infectious disease detection to precision oncology. Backed by deep technical expertise and global manufacturing capabilities, we deliver consistent quality, rapid turnaround and scalable supply—empowering diagnostic innovators to accelerate development and bring products to market with confidence.
• Optimized master mixes for LAMP and qPCR • Nucleotides and other supporting reagents
www.meridianbioscience.com/lifescience
HOW MERIDIAN’S PCR CHEMISTRY COMPARED IN TARGETED NGS INSIGHTS FROM A DIAGNOSTIC DEVELOPER’S STUDY
The study aimed to understand how differences in enzyme formulation and amplification chemistry could impact key performance factors such as: • Amplicon coverage uniformity, especially in areas of high GC-content • Variant detection sensitivity and specificity of low- frequency BRCA1/2 variants (below 1%) • Compatibility with challenging samples such as low- input ctDNA and degraded gDNA By systematically comparing amplification efficiency and sequencing quality, the developer assessed how different enzyme chemistries impacted the generation of high- quality sequencing libraries for its tNGS assay.
A diagnostic developer with a commercial tNGS assay for BRCA1 and BRCA2 variant detection conducted a study to compare its current enzyme chemistry with Meridian’s as part of an exploratory effort to identify alternative solutions compatible with their platform and workflow. For the evaluation, the customer selected Meridian’s Lyo-Ready Direct DNA qPCR Blood Mix [MDX122] to test alongside its existing mix in an established hereditary breast cancer screening assay. The workflow used an amplicon-based tNGS approach featuring highly-multiplexed PCR to amplify 177 target regions prior to sequencing.
STUDY WORKFLOW
1 Sample
Preparation
Plasma samples from breast cancer patients were processed to extract circulating tumor DNA (ctDNA) using methods optimized for low-input and fragmented DNA. DNA quality and quantity were confirmed with Qubit and Agilent TapeStation. HORIZON DNA standards were included for consistency, and water served as a no-template control (NTC).
2 PCR & Library Preparation
A two-step multiplex PCR was performed to amplify 177 BRCA targets in triplicate. The first step used gene-specific primers; the second added adapters and barcodes for multiplexing. The commercial enzyme mix was replaced with Meridian’s mix, while all other PCR steps remained unchanged. Gel electrophoresis confirmed clean amplification. Libraries then underwent bead clean-up, size selection, quantification and adapter ligation for sequencing readiness.
3 Sequencing & Analysis
Sequencing was performed on a DNBSEQ-G400 platform (PE100 mode), delivering paired-end reads for high-resolution analysis. Data evaluation included mapping to GRCh38, variant calling (GATK, VarDict), amplicon coverage assessment, GC-bias analysis and error rate checks to confirm accurate detection of low-frequency variants.
www.meridianbioscience.com/lifescience
APPLICATION NOTE qPCR MIXES FOR tNGS
STUDY RESULTS
The study assessed two PCR chemistries in a hereditary breast cancer assay to determine performance across critical sequencing metrics. Using a two-step, 177-target multiplex qPCR approach, BRCA1 and BRCA2 regions were amplified with Meridian’s Lyo-Ready Direct DNA qPCR Blood Mix [MDX122] and a commercial alternative. After library preparation, sequencing on the DNBSEQ-G400 platform showed that both mixes performed strongly, with mapping rates exceeding 99% and coverage uniformity above 85%. These results confirm that Meridian’s optimized chemistry enables reliable amplification and well-balanced libraries—essential for accurate variant detection and consistent performance in tNGS workflows.
The results showed equivalent performance across all critical metrics, confirming that a mix validated for qPCR can also power complex tNGS workflows . For developers, this demonstrates the versatility of Meridian’s chemistry: one mix, multiple applications—helping simplify validation, reduce risk and streamline assay development pipelines. Meridian’s Lyo-Ready Direct DNA qPCR Blood Mix delivered uniform amplification and supported accurate detection of BRCA1 and BRCA2 variants— even at low frequencies from low-input DNA .
KEY SEQUENCING OUTCOMES
Meridian’s Mix Commercial Mix
MAPPING RATE
99.9% 99.9% 99.9% 99.6%
TARGET RATE
UNIFORMITY
86.7% 88.6%
0%
20%
40%
60%
80%
100%
TARGETED NGS (tNGS) A GROWING FORCE IN PRECISION DIAGNOSTICS Targeted next-generation sequencing (tNGS) is rapidly becoming a cornerstone of precision medicine, bridging the gap between single-target assays and genome-wide approaches. It offers high multiplexing while maintaining
manageable data complexity. Clinical practice now relies heavily on tNGS, with many FDA- approved commercial assays for solid tumors, hematologic malignancies and liquid biopsy applications widely adopted in both centralized and specialty oncology labs. 3 This growing clinical use is fueling significant market expansion—the global NGS market is projected to rise from $12.9 billion in 2023 to nearly $98 billion by 2035. 4
With FDA-approved tests already in use and a market projected to hit $98B by 2035 , tNGS is transforming precision diagnostics .
EXAMPLES OF FDA-CLEARED TARGETED NGS ASSAYS USED IN CLINICAL PRACTICE
APPLICATION NOTE qPCR MIXES FOR tNGS
To support amplicon-based tNGS workflows, Meridian provides a modular portfolio of high-performance, glycerol-free reagents—including optimized mixes, enzymes and nucleotides—engineered for sensitive amplification, efficient clean-up and seamless integration into your workflow. From PCR through library preparation , our solutions are designed to reduce hands-on time, improve assay consistency and deliver reliable results at scale. FROM PCR TO LIBRARY PREP REAGENTS ENGINEERED FOR RELIABILITY
Here’s How Meridian Fits into Your tNGS Workflow:
TO LEARN MORE:
REFERENCES 1. Zheng, Y. R., et al. (2024). Comparison of targeted next-generation sequencing and metagenomic next-generation sequencing in the identification of pathogens in pneumonia after congenital heart surgery: a comparative diagnostic accuracy study. Italian Journal of Pediatrics , 50 (1), 174. https://doi.org/10.1186/s13052-024-01749-z 2. Broude, N. E., et al . (2001). Multiplex allele-specific target amplification based on PCR suppression. Proc Natl Acad Sci USA , 98 (1), 206-211. https://doi.org/10.1073/pnas.98.1.206 3. Merino, D. M., et al. (2020). Establishing guidelines to harmonize tumor mutational burden (TMB): In silico assessment of variation in TMB quantification across diagnostic platforms: phase I of the Friends of Cancer Research TMB Harmonization Project. Journal for ImmunoTherapy of Cancer, 8, e000147. https://doi.org/10.1136/ jitc-2019-000147 4. Allied Market Research, Next Generation Sequencing Market Forecast, 2023-2035, April 2024.
Discover how Meridian’s molecular solutions can help optimize your targeted NGS workflows. From high-performance PCR chemistries to custom formulations and scalable manufacturing, we provide the tools and expertise to accelerate your assay development. VISIT: meridianbioscience.com/lifescience/ precision-oncology-solutions
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