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.
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