A robust targeted sequencing approach for low input and variable quality DNA from clinical samples.

Area of Science:

• Molecular Biology
• Genomics
• Cancer Research

Background:

• Next-generation sequencing (NGS) gene panels are crucial for identifying actionable mutations in cancer diagnostics.
• Clinical samples, particularly formalin-fixed, paraffin-embedded (FFPE) specimens, often yield low-quality, degraded DNA.
• Current NGS assays frequently require extensive PCR amplification, introducing bias and artifacts.

Purpose of the Study:

• To evaluate a targeted sequencing assay using Oligonucleotide Selective Sequencing (OSS) for analyzing low-quality DNA.
• To address the need for a reliable method that minimizes PCR amplification for cancer mutation detection.
• To assess the assay's performance with limited and damaged DNA input from clinical samples.

Main Methods:

• Utilized Oligonucleotide Selective Sequencing (OSS) for targeted gene enrichment and variant identification.
• Employed a DNA repair process optimized for FFPE samples, followed by single-stranded DNA adaptor ligation.
• Incorporated a primer-based capture technique, reducing reliance on extensive PCR amplification.

Main Results:

• Achieved high read coverage uniformity across a 130-gene panel with as little as 10 ng of input DNA.
• Demonstrated accurate detection of single nucleotide variants (SNVs) and insertion/deletion (indel) mutations.
• Enabled reliable assessment of copy number alterations (CNAs) alongside SNV and indel detection.
• Successfully identified sequence variants in DNA from diverse matched clinical FFPE samples.

Conclusions:

• The OSS-based targeted sequencing assay effectively analyzes low-quantity, degraded DNA from FFPE samples.
• This method provides high-fidelity libraries with reduced PCR bias, improving diagnostic accuracy for cancer mutations.
• The assay facilitates accurate detection of SNVs, indels, and CNAs, offering a valuable tool for clinical cancer genomics.