CONCLUSION: Optimizing Enzyme Chemistry for Reliable tNGS Workflows
This study evaluated the impact of different enzyme chemistries on multiplex PCR amplification on targeted NGS (tNGS) workflow, comparing Meridian’s Lyo-Ready Direct DNA qPCR Blood Mix (MDX122) with a commercial alternative in the context of BRCA1 and BRCA2 variant detection. The results demonstrated that enzyme chemistry plays a crucial role in achieving uniform amplification, minimizing bias, and ensuring high-confidence variant detection, particularly in low-input and degraded DNA samples. By employing a robust two-step multiplex PCR approach using a BRCA panel with 177 primer pairs, Meridian’s qPCR mix achieved high specificity and yield in amplifying BRCA1 and BRCA2 gene regions. Gel electrophoresis confirmed that the PCR products were of the expected size, with distinct bands, underscoring the accurate and reliable multiplexing capability of Meridian’s qPCR formulation. The subsequent sequencing on the DNBSEQ-G400 platform further validated the high quality of the amplified fragments, with both Meridian’s mix and the commercial tNGS kit displaying mapping rates exceeding 99% and target region coverage uniformity above 85%. Moreover, the observed allele frequencies closely aligned with theoretical expectations, confirming the precision and reproducibility of the tNGS workflow.
These findings support the broader application of optimized qPCR formulations in tNGS workflows, particularly for hereditary cancer screening and liquid biopsy-based oncology applications. The exceptional specificity and multiplexing capability demonstrated by Meridian’s qPCR mix reinforces its utility in high-sensitivity genetic analysis, ensuring high-quality sequencing outcomes for variant detection in challenging sample types. Future studies could further expand on these results by evaluating additional gene targets, sequencing platforms,
and real-world clinical sample sets, paving the way for more reliable and accurate diagnostic workflows in
precision medicine.
By employing a robust two-step multiplex PCR approach using a BRCA panel with 177 primer pairs, Meridian’s qPCR mix achieved high specificity and yield in amplifying BRCA1 and BRCA2 gene regions.
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