Study Design: Evaluating PCR Efficiency and tNGS Performance
> Compatibility with challenging sample types – Testing performance in low-input and fragmented DNA conditions. By systematically evaluating amplification efficiency and sequencing performance, this study assesses the efficacy of a novel qPCR mix Lyo-Ready Direct DNA qPCR Blood (MDX122) within a multiplex PCR-based tNGS workflow. It investigates how enzyme formulation and PCR optimization influence amplification uniformity, specificity, and overall sequencing outcomes to enhance the reliability and accuracy of tNGS workflows.
This study applies a two-step multiplex PCR strategy to amplify BRCA1 and BRCA2 gene regions, evaluating its performance in a tNGS-based hereditary breast cancer screening workflow. The study compares Meridian’s Lyo-Ready Direct DNA qPCR Blood (MDX122) with a commercial tNGS kit with a customized panel (ordered from https://atoplex.mgi-tech.com/) to assess: > Amplicon coverage uniformity – Ensuring balanced representation of all target loci across sequencing reads. > Specificity and sensitivity of variant detection – Evaluating detection accuracy for low-frequency BRCA1/2 mutations in ctDNA samples.
Materials and Methods Sample Preparation and DNA Standards
PCR cleanup and library preparation steps included: > Magnetic bead purification of amplicons to remove unincorporated primers and reaction components. > Size selection and quantification using the Agilent Bioanalyzer and Qubit to ensure that the final DNA fragments were within the optimal sequencing range. > Adapter ligation and indexing to enable sample multiplexing during sequencing. Next-Generation Sequencing and Data Analysis Following PCR and library preparation, sequencing was performed on the DNBSEQ-G400 platform in PE100 mode, generating paired-end reads. The sequencing performance was assessed based on raw and clean reads, mapping efficiency, and target region coverage. Data analysis included: > Read Alignment – Mapping to the GRCh38 human reference genome to ensure high sequencing accuracy. > Variant Calling – Detection of BRCA1 and BRCA2 mutations using industry-standard pipelines (GATK, VarDict). > Amplicon Coverage Analysis – Assessing uniformity, dropout rates, and bias across GC-rich regions to evaluate amplification consistency. > Error Rate and Background Noise Evaluation – Ensuring high-confidence variant calling, particularly for low-frequency mutations in plasma genomic DNA (gDNA) samples.
HORIZON DNA standards were used as the template DNA to ensure consistency in the evaluation of BRCA1 and BRCA2 amplification. Water was included as a no-template control (NTC) to assess potential contamination. In a separate analysis, plasma samples were collected from breast cancer patients, and circulating tumor DNA (ctDNA) was extracted. The extraction process was optimized to maximize recovery of low-input and fragmented DNA, ensuring compatibility with tNGS-based liquid biopsy applications. DNA quantity and quality were assessed using fluorometric quantification (Qubit) and fragment analysis (Agilent TapeStation) to confirm
suitability for downstream PCR amplification. Multiplex PCR and Library Preparation
A two-step multiplex PCR approach was employed to amplify the BRCA1 and BRCA2 gene regions using a BRCA panel consisting of 177 primer pairs. The PCR protocol consisted of an initial 10 cycles to enrich low-input DNA, followed by 19 additional cycles to optimize amplification. The study compared two conditions: the experimental group using Meridian’s Lyo-Ready Direct DNA qPCR Blood (MDX122) and the control group using the commercial tNGS kit. Triplicate reactions were performed for each condition to ensure reproducibility. PCR amplification efficiency and specificity were evaluated through gel electrophoresis, confirming the presence of distinct and expected bands for both groups while ensuring that no amplification was observed in the NTC samples.
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