Benchmarking Q40 sequencing for sensitive and efficient detection of rare genomic variants

BACKGROUND: Phred quality score (Q score) is critical for sequencing accuracy, yet the impact of Q40-achieving sequencing technologies (99.99% accuracy) on detecting subtle biological variations remains unvalidated. RESULTS: Using a comprehensive set of well-established DNA/RNA reference materials (Quartet, NIST-RM8398, SEQC2-HCC1395/BL, MAQC, and ERCC), we benchmarked Q40 sequencing (Element AVITI) against the conventional Q30 standard (Illumina NovaSeq 6000). Q40 reduced required sequencing depth by 33.3% while maintaining accuracy for germline variants (20 × vs. 30 ×) and somatic single-nucleotide variant/insertion-deletion (SNV/InDel) (60 × vs. 90 ×). Crucially, Q40 enhanced sensitivity for low-frequency somatic mutations (variant allele frequency, VAF ≤ 0.2) by 33.3% and sixfold higher copy number variation (CNV) detection reproducibility (60.3% vs. 10.4%) with Q40 at 30 × depth, directly reducing per-sample volumes by 33.3-60% and theoretically reducing sequencing costs by 2.2-31.7%. In addition, Q40 improved the discriminatory resolution between biological samples with 13.1% signal-to-noise ratio (SNR) enhancement. CONCLUSIONS: Taken together, our findings establish the value of Q40 sequencing as a sensitive and cost-effective method for low-frequency variant detection. While this positions it as a promising tool for precision oncology, its performance in real-world clinical applications remains to be evaluated in future studies.