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
Powered by FlippingBook