Understanding the Technological Shift in NGS

Recent advancements in genomic research have significantly improved mutation detection. However, researchers have identified significant NovaSeq 6000 sequencing errors that may compromise the accuracy of somatic variant detection. This issue primarily stems from Illumina’s transition from the four-color chemistry found in the HiSeq series to a two-color fluorescent dye chemistry in the NovaSeq series. While this change enhances sequencing speed and scalability, it introduces a recurrent artifact that can lead to false-positive results in specific clinical contexts.

The Impact of NovaSeq 6000 Sequencing Errors on Clinical Diagnostics

A recent study re-analyzed whole-genome sequencing experiments and revealed that the NovaSeq 6000 platform shows a higher frequency of T-to-G and A-to-C substitutions. Although the overall per-base error rate remains low, these artifacts significantly impact the identification of low variant allele frequency (VAF) somatic mutations. Specifically, mutations with low read support in high-depth samples are the most vulnerable to these errors. This finding is particularly critical for oncology and hematology studies involving mosaic mutations or liquid biopsies where a matched normal tissue sample is often unavailable.

The research demonstrated that these artifactual variant calls occur disproportionately at NT[TG] trinucleotides. Consequently, clinicians must exercise caution when interpreting T-to-G variants found in these specific genomic environments. Fortunately, laboratories can use bioinformatic strategies to mitigate these effects. By leveraging the specific trinucleotide context, researchers can filter out the excess T-to-G calls from their datasets. Therefore, a deep understanding of these chemistry-driven biases is essential for ensuring the reliability and clinical actionability of next-generation sequencing reports.

FAQ

Why do these errors occur on the NovaSeq 6000?

The NovaSeq 6000 platform utilizes a two-color chemistry system to improve efficiency. In this setup, the nucleotide Guanine (G) is detected when no fluorescent signal is present. Consequently, if a signal is lost or obscured during a sequencing cycle, the instrument may incorrectly call a Guanine, leading to T-to-G or A-to-C artifacts.

How do these sequencing artifacts impact clinical oncology results?

These errors predominantly affect low-frequency variants, which are vital for liquid biopsies and detecting minimal residual disease. Such artifacts can mimic pathogenic mutations in genes like TP53 or KIT, potentially leading to inaccurate tumor mutational burden (TMB) assessments and misinformed clinical decisions.

Disclaimer: This content is for informational and educational purposes only. It does not constitute medical advice or a substitute for professional clinical judgment. Diagnostic and treatment decisions should be based on a comprehensive evaluation of the patient and the specific laboratory context. Refer to the latest local and national guidelines for clinical practice.

References

Fu BJ et al. A recurrent sequencing artifact on Illumina sequencers with two-color fluorescent dye chemistry and its impact on somatic variant detection. Genome Biol. 2026 Apr 17. doi: 10.1186/s13059-026-04081-3. PMID: 41998753.

Fu BJ, et al. Systematic artifacts from Illumina two-color chemistry confound variant identification and actionability in clinical panels. medRxiv. 2025 Oct 20. doi: 10.1101/2025.10.17.25338254.