qPCR validation of next generation sequencing results

qPCR validation of next-generation sequencing (NGS) results remains critical for ensuring data accuracy, resolving technical limitations, and meeting publication or clinical standards. While NGS enables high-throughput genomic analysis, qPCR provides targeted verification through its simplicity, sensitivity, and cost-effectiveness, particularly for low-abundance targets or clinically actionable variants.

Key Reasons for qPCR Validation

  1. Error Mitigation:
    NGS workflows involve complex sample preparation, bioinformatics pipelines, and potential biases (e.g., GC-rich region underrepresentation)[1]. qPCR reduces false positives/negatives by independently confirming variants or expression levels[2][3].
  2. Sensitivity for Low-Abundance Targets:
    qPCR outperforms NGS in detecting low-copy transcripts or rare variants (<1% allelic frequency) due to its linear amplification and lack of reliance on sequencing depth[1][3].
  3. Reference Genome Limitations:
    NGS aligns reads to error-containing reference genomes, which may disregard SNPs or structural variations. qPCR uses actual templates, bypassing alignment biases[1].

Validation Workflow Integration

  • Orthogonal Verification:
    qPCR validates NGS-identified variants or differentially expressed genes. For example, COVID-19 variant tracking combined NGS with RT-qPCR assays targeting spike protein deletions, achieving >95% concordance[4].

 

Method     Role in NGS Validation     Key Advantage
qPCR    Target-specific verification    Cost-effective, high-throughput

 

Best Practices

  • Assay Design:
    Use TaqMan probes or FRET-based assays with in silico/in vitro validation to ensure primer specificity[4][5].
  • Data Normalization:
    Address NGS assumptions (e.g., uniform mRNA levels across samples) by validating housekeeping genes via qPCR[1][6].
  • Guideline Compliance:
    Follow Association of Molecular Pathology (AMP) recommendations for analytical validation, including reference materials and error-based quality controls[6].

Complementary Use Cases

  • Upstream QC: Verify cDNA integrity pre-NGS using qPCR[2].
  • Downstream Prioritization: Narrow NGS-derived gene lists to clinically relevant targets for high-confidence validation[3][5].

While emerging technologies like ddPCR enhance precision, qPCR remains indispensable for balancing NGS scalability with rigorous validation, particularly in clinical diagnostics and peer-reviewed research[1][6][2].

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  1. https://sciencellonline.com/blog/value-of-qpcr-in-the-next-generation-sequencing-era/
  2. https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/ngs-comparison.html
  3. https://www.illumina.com/science/technology/next-generation-sequencing/beginners/advantages/ngs-vs-qpcr.html
  4. https://pmc.ncbi.nlm.nih.gov/articles/PMC10059866/
  5. https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-applications/gene-expression-using-real-time-pcr/verification-ngs-microarray-results.html
  6. https://pmc.ncbi.nlm.nih.gov/articles/PMC6941185/