Meridian is a primary manufacturer of specialized high-quality molecular reagents and offers solutions to a wide range of industries to diagnose and treat diseases, discover new therapeutics or develop tests for environmental, food and cosmetic safety.
CASE STUDY
A Performance Evaluation of BRCA1/2 Amplification in a Breast Cancer Screening Workflow Optimizing Multiplex PCR Strategies for Targeted NGS (tNGS)
INTRODUCTION: Targeted NGS for Clinical Applications
Despite these advantages, the accuracy and efficiency of tNGS workflows rely on optimized amplification strategies, particularly in multiplex PCR-based approaches. Challenges such as primer-dimer formation, GC-content variability, and polymerase efficiency can impact amplification uniformity and specificity. 2 To improve assay reliability, minimizing amplification bias requires optimized enzyme chemistry. This study evaluates the performance of Meridian’s Lyo-Ready Direct DNA qPCR Blood (MDX122) within a tNGS workflow to determine its impact on amplification efficiency, specificity, and sequencing quality. The study specifically assesses BRCA1 and BRCA2 variant detection in a breast cancer screening application, using a highly multiplexed PCR approach followed by targeted sequencing. By comparing Meridian’s qPCR mix to a commercially available alternative, the study aims to provide insights into how enzyme formulation and amplification chemistry influence tNGS performance, particularly in detecting low-frequency variants from plasma genomic DNA (gDNA).
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. To address these limitations, Targeted NGS (tNGS) has emerged as a cost-effective, high-sensitivity alternative, focusing sequencing efforts on specific genes or genomic regions. By enriching for targeted panels, tNGS enables deeper sequencing coverage, improves variant detection sensitivity, and lowers overall sequencing costs. 1
Targeted NGS (tNGS) has emerged as a cost-effective, high-sensitivity alternative
www.meridianbioscience.com/lifescience
1
BACKGROUND: The Role of tNGS in Hereditary Cancer Screening
tNGS is widely used in oncology, hereditary disease screening, and precision medicine for its ability to achieve ultra-deep sequencing, often exceeding 10,000× coverage. Its high sensitivity is ideal for detecting low-frequency somatic mutations in oncology and liquid biopsy applications, making it a key tool for minimal residual disease (MRD) monitoring and early cancer detection.
tNGS relies on two main enrichment methods: > Hybridization Capture: Uses biotinylated probes to bind and isolate target sequences. It is highly specific but slower. > Multiplex PCR-Based Enrichment: Uses multiple primers to amplify target regions quickly with minimal DNA input. However, it can introduce amplification bias and variable coverage if not optimized.
Figure 1: NGS Library Prep Workflows
Whole Genome Sequencing (WGS)
Fragmentation
End-Repair
Adapter Ligation
Lib. Amplification
Library QC
Adapter Ligation
Amplicon-based Targeted NGS
Target Amp.
Library QC
OR
Indexing Amp.
Hybrid-Capture Targeted NGS
Adapter Ligation
Lib. Amplification
Fragmentation
End-Repair
Hybrid Capture Indexing Amp.
Library QC
Figure 1. WGS fragments the entire genome, providing comprehensive coverage and high sequencing depth. In contrast, tNGS takes a more focused approach, either through hybrid capture, where biotinylated probes bind to hundreds to thousands of specific targets (e.g., 500–50,000), or through an amplicon-based method that uses multiplex PCR to amplify ten to a few hundred targets (e.g., 10–500) before sequencing.
> Amplification Bias and Uniformity: Uneven amplification across target regions can result in low sequencing coverage for certain amplicons, affecting variant detection sensitivity. > Low-Input DNA Compatibility: Clinical samples, especially circulating tumor DNA (ctDNA) and formalin-fixed, paraffin- embedded (FFPE) samples, often contain degraded, fragmented, or low-concentration DNA, requiring robust amplification strategies. > Polymerase Fidelity and Efficiency: Enzyme chemistry must support high processivity and error correction to reduce
For hereditary cancer mutations, such as BRCA1 and BRCA2, multiplex PCR-based tNGS is often preferred due to its higher sensitivity, faster turnaround time, and cost-effectiveness compared to hybridization-based methods. 3 It requires less input DNA, achieves higher on-target efficiency ( ~ 90–95%), and provides more uniform coverage, especially in high-GC regions. Additionally, amplicon sequencing excels at detecting small variants, such as SNVs and indels, making it ideal for routine hereditary cancer screening. Challenges in Multiplex PCR Amplification for tNGS Workflows Minimizing amplification bias requires optimized enzyme chemistry to improve assay reliability. Multiplex PCR is widely used to enrich targeted regions, but achieving uniform, high-yield amplification can be challenging for several reasons: > Primer Design and Specificity: Poorly designed primers can cause off-target amplification, primer-dimer formation, and GC bias, leading to coverage gaps and reduced sequencing accuracy.
sequencing artifacts and improve variant calling accuracy.
2
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.
www.meridianbioscience.com/lifescience 3
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.
4
Meridian’s qPCR mix outperformed the competitor—delivering reliable library prep from just 5 ng of gDNA, with fewer failures, higher fidelity, and cleaner sequencing results.
Figure 3: Sequencing Results from Amplified DNA Fragments A) SEQUENCING PERFORMANCE
Raw_reads
Clean_reads
Map_rate (%)
Targeted_rate (%)
Uniformity (>0.1x)
41,044,434
40,966,416
99.8
99.8
85.8
Commercial tNGS Kit
36,644,668
36,578,418
99.9
99.8
87.8
35,911,938
35,851,470
99.9
99.8
86.6
15,056,954
14,970,350
99.9
99.96
87.3
Direct DNA qPCR Blood Mix (MDX122)
23,806,042
23,710,302
99.9
99.7
88.7
16,411,848
16,312,476
99.9
99.6
89.7
B) VARIANT DETECTION
Lyo-Ready Direct DNA qPCR Blood Mix (MDX122)
Targeted Gene
cds
pos
Theoretical
Commercial tNGS Kit
BRACA1
c.3548A>G
17:43091983
7.5%
8.0%
7.8%
BRACA1
c.2458A>G
17:43092919
7.5%
8.8%
8.6%
BRACA1
c.2612C>T
17:4309291
15.0%
15.0%
14.3%
BRACA1
c.4327C>T
17:43082434
32.5%
32.8%
33.0%
BRACA1
c.4900A>G
17:43071077
32.5%
7.5%
7.6%
BRACA1
c.4258G>T
13:32338613
10.0%
31.3%
31.7%
BRACA2
c.8021dup
13:32363217
32.5%
8.3%
8.4%
BRACA2
c.5073del
13:32339421
40.0%
32.1%
32.6%
BRACA2
c.5351del
13:32339699
7.5%
37.0%
37.5%
BRACA2
c.86SA>C
13:32332343
7.5%
7.2%
7.3%
BRACA2
c.2971A>G
13:32337326
7.5%
6.6%
7.0%
BRACA2
c.7397T>C
13:3235525
100.0%
100.0%
99.6%
HORIZON BRCA Somatic Multiplex l gDNA Multiplex
Taken together, these findings confirm that Meridian’s Lyo-Ready Direct DNA qPCR Blood Mix (MDX122) effectively amplifies targeted DNA with high specificity and yield, producing high-quality sequencing libraries even from challenging sample types. The mix ensures reliable variant detection, particularly for low-frequency mutations, without compromising performance in a tNGS workflow. Figure 3. A) Sequencing reads of the commercial tNGS kit vs Meridian’s Lyo-Ready Direct DNA qPCR Blood Mix (MDX122); B) Variant detection in both groups. Both mixes show similar sequencing metrics with high mapping rates (>99%) and uniformity (>85%), reflecting consistent data quality. The observed allele frequencies in both mixes closely align with theoretical values for all variants, demonstrating accurate variant detection across experiments. Raw Reads (Raw_reads): Total raw reads generated from sequencing. Clean Reads (Clean_reads): Reads retained after filtering for quality. Mapping Rate (Map_rate%): Percentage of reads successfully mapped to the reference genome. Target Rate (Target_rate%): Percentage of reads aligned to the target regions. Uniformity (>0.1x): Measure of how evenly sequencing reads cover the target regions. CDS (Coding Sequence): Variants in the coding sequence. POS (Position): Genomic position of each variant. Theoretical: Expected allele frequency of the variant. Commercial tNGS Kit: Measured allele frequency in the control group. Lyo-Ready Direct DNA qPCR Mix (MDX122): Measured allele frequency in the experimental group. These findings validate that Meridian’s Lyo-Ready Direct DNA qPCR Blood (MDX122) provides precise and consistent amplification, supporting high-confidence variant mix detection in a targeted sequencing approach.
www.meridianbioscience.com/lifescience
5
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.
REFERENCES:
1. Zheng YR, Chen XH, Chen Q, Cao H. 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. Ital J Pediatr. 2024 Sep 12;50(1):174. doi: 10.1186/s13052-024-01749-z. PMID: 39267108; PMCID: PMC11395185. 2. Broude NE, Zhang L, Woodward K, Englert D, Cantor CR. Multiplex allele-specific target amplification based on PCR suppression. Proc Natl Acad Sci U S A. 2001 Jan 2;98(1):206-11. doi: 10.1073/pnas.98.1.206. PMID: 11136256; PMCID: PMC14569. 3. Singh RR. Target Enrichment Approaches for Next-Generation Sequencing Applications in Oncology. Diagnostics (Basel). 2022 Jun 24;12(7):1539. doi: 10.3390/diagnostics12071539. PMID: 35885445; PMCID: PMC9318977.
6
www.meridianbioscience.com/lifescience 7
About Meridian
Meridian is a fully integrated life science company that develops, manufactures, markets, and distributes a broad range of innovative diagnostic products and critical raw materials. We are dedicated to developing and delivering better solutions that give answers with speed, accuracy, and simplicity that redefine the possibilities of life from discovery to diagnosis. As the Life Science division of Meridian, our focus is on supporting immunological and molecular test manufacturers with original raw materials for human, animal, plant, and environmental applications. The large portfolio of antigens, antibodies, blockers, molecular enzymes, nucleotides, and optimized mixes for qPCR and isothermal amplification applications are designed to simplify assay design and enable accurate test results. We strive to provide our customers with solutions they need when they need them – from novel antigens and antibodies to major disease outbreaks such as Zika and SARS-CoV-2 to pioneering the market with our innovative air-dried qPCR/RT-qPCR mixes. We take pride in providing our customers with unparalleled support, customer service, and quality.
Authors
Valerie Midgley, Ph.D. Global Marketing Manager, Campaigns & Content LIFE SCIENCE
Alpha Chen Senior Product Specialist LIFE SCIENCE
Ordering information:
USA 5171 Wilfong Road Memphis, Tennessee 38134 Phone: +1 901-382-8716 Fax: +1 901-333-8223
Email: info@meridianlifescience.com Orders: orders@meridianlifescience.com www.meridianbioscience.com/lifescience
Connect with us:
ISO 13485 Certified
04/25
Page 1 Page 2 Page 3 Page 4 Page 5 Page 6 Page 7 Page 8Powered by FlippingBook