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bioRxiv · 10.1101/2025.08.19.671033

Validation of the NovaSeq6000 Platform and automated library preparation for CE-IVD equivalence

Pasquinelli, E.·Rollo, G.·Tinella, F.·Danelli, M.·Minetto, S.·Casamassima, G.·Bader, M.·Calonaci, L.·Colavecchio, O. L.·Tita, R.·Mancini, R.·Baldassarri, M.·Lo Rizzo, C.·Pinto, A. M.·Ariani, F.·Fallerini, C.·Montagna, P.·Mezzatesta, V.·Bruttini, M.·Renieri, A.
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Abstract

The implementation of next-generation sequencing (NGS) technologies in clinical diagnostics requires rigorous validation of sequencing platforms and analytical workflows. In this study, we validated the performance of the Illumina NovaSeq6000 Research Use Only (RUO) platform, combined with automated library preparation using the Hamilton Microlab STAR system, by comparison to the CE-IVD certified NovaSeq6000Dx platform, which currently relies on manual library preparation. A total of 96 clinical samples underwent whole-exome sequencing (WES) on both platforms. Variant detection performance was assessed for single nucleotide variants (SNVs) and copy number variants (CNVs). The RUO platform demonstrated 100% concordance with the CE-IVD system for clinically relevant SNVs, with full agreement across positive, negative, and overall percent agreement metrics. For CNVs larger than 150 kb, the positive percent agreement was 79%, rising to 91.7% for CNVs >900 kb. Coverage uniformity and autosomal callability were consistently high across platforms. These results confirm the analytical equivalence of the NovaSeq6000 RUO configuration with automated library preparation for clinical-grade WES. This validation framework supports the adoption of scalable, cost-effective workflows that can achieve diagnostic performance comparable to CE-IVD certified systems and may facilitate routine implementation of exome sequencing in clinical laboratories. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/671033v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1735683org.highwire.dtl.DTLVardef@134d50dorg.highwire.dtl.DTLVardef@18f6e07org.highwire.dtl.DTLVardef@180a3e8_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Pasquinelli, E., Rollo, G., Tinella, F., Danelli, M., Minetto, S., Casamassima, G., Bader, M., Calonaci, L., Colavecchio, O. L., Tita, R., Mancini, R., Baldassarri, M., Lo Rizzo, C., Pinto, A. M., Ariani, F., Fallerini, C., Montagna, P., Mezzatesta, V., Bruttini, M., Renieri, A.. 2025-08-23. Validation of the NovaSeq6000 Platform and automated library preparation for CE-IVD equivalence. https://doi.org/10.1101/2025.08.19.671033

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