RESULTS: PCR Performance and Amplicon Coverage Uniformity
The comparison between the two PCR enzyme chemistries revealed that Meridian’s qPCR mix provided more consistent coverage across all targeted regions (Lyo-Ready Direct DNA qPCR Blood, MDX122), with a lower incidence of amplicon dropout in high-GC-content regions. The coefficient of variation (CV) for coverage depth was significantly reduced in samples amplified with Meridian’s mix, indicating less amplification bias compared to the commercial alternative. Specifically, PCR product concentrations were measured across both qPCR formulations. The control group, amplified using the commercially available tNGS kit, produced concentrations ranging from 24.4–28.2 ng/ µ L, while the experimental group, amplified using Meridian Lyo-Ready Direct DNA qPCR Blood (MDX122), demonstrated slightly lower concentrations between 14.9–16.3 ng/ µ L. The results revealed a high degree of consistency and no amplification was detected in the No Template Control (NTC) samples, confirming the absence of contamination. The findings were further validated through gel electrophoresis analysis (Figure 2). Distinct and expected PCR bands were observed for both Meridian’s mix and the commercial tNGS kit, with no bands present in the NTC samples, confirming the specificity of the amplification. Meridian’s mix exhibited slightly
lower band intensity, which correlates with the lower PCR product concentrations previously measured. Despite this difference in yield, the amplicon sizes remained consistent across both groups, demonstrating that the Meridian qPCR mix achieved accurate and specific amplification, ensuring the reliability of subsequent sequencing analysis.
Figure 2: Gel electrophoresis of PCR amplification products
Figure 2. Results show that both PCR chemistries generated the expected PCR products with high specificity, as evidenced by the absence of non-specific bands in the NTC lanes. Lanes 1-3: Control group amplified using the commercial tNGS kit; Lane 4: No template control (NTC) for the control group; Lanes 5-7: Experimental group amplified using Meridian Lyo-Ready Direct DNA qPCR Blood (MDX122); Lane 8: NTC for the experimental group.
Distinct and expected PCR bands were observed for both the control and experimental groups, with no bands present in the NTC samples, confirming the specificity of the amplification.
Sequencing & Variant Detection Performance The sequencing performance and variant detection results (Figure 3) demonstrated high consistency between the control and experimental groups, confirming the robustness of both PCR chemistries in targeted NGS workflows. Sequencing was performed on the DNBSEQ-G400 platform in PE100 mode, generating comparable numbers of raw and clean reads across both groups. Mapping rates exceeded 99%, and target region coverage uniformity remained above 85%, highlighting even sequencing depth across all targeted loci. These metrics reflect a high-quality sequencing output and indicate that both chemistries produced well-balanced libraries suitable for downstream variant analysis. Variant detection sensitivity and specificity were also comparable between both chemistries, with Meridian’s qPCR
mix yielding slightly higher variant calling confidence, particularly in low-input ctDNA samples. The detection of low-frequency BRCA1/2 variants was enhanced in samples amplified using Meridian’s mix, particularly for mutations with allele frequencies below 1%. This improvement is likely due to more efficient amplification of genomic DNA (gDNA), resulting in better representation of minor allele variants. Additionally, the compatibility of each mix with low-input and degraded DNA was assessed. Meridian’s qPCR mix exhibited greater robustness in low-DNA conditions, enabling successful library preparation from as little as 5 ng of gDNA, whereas the commercial alternative showed higher failure rates at inputs below 10 ng. Furthermore, higher polymerase fidelity and processivity contributed to reduced background noise and off-target artifacts, improving overall sequencing accuracy.
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