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Evaluation of iconPCR™ Technology for Normalization of truCOVER® DNA Libraries from FFPE DNA

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Abstract

Formalin-Fixed Paraffin-Embedded (FFPE) samples serve as the gold standard for clinical tissue preservation, enabling long-term maintenance of tissue architecture and providing a vast resource for retrospective pathology studies. However, NGS analysis of FFPE material is frequently compromised by nucleic acid degradation and residual chemical cross-linking, which impede library preparation and library amplification efficiency. More importantly, DNA amounts from FFPE samples are often limited, such that libraries need to be amplified by PCR. Since amplification efficiency varies with the initial DNA quality, picking the right amplification cycle number is not intuitive. To address these challenges, we investigated the combination of the Covaris truCOVER DNA Library workflow and n6’s iconPCR to agnostically amplify FFPE DNA libraries to a common yield, sufficient for pooling for Illumina NGS. The standard protocol for high quality genomic DNA specifies a 0.1 to 500 ng input range and 10 to 500 ng for FFPE DNA. We amplified libraries with inputs ranging from 1 to 50 ng DNA varying significantly in quality and used n6’s iconPCR technology to normalize library yields for direct pooling for NGS analysis. The iconPCR platform’s proprietary AutoNorm™ feature facilitates the adaptive determination of optimal cycle numbers for individual samples in real-time. By integrating truCOVER DNA Library prep and iconPCR-PCR based amplification using truCOVER DNA Library Amplification kit into one continuous workflow we successfully pooled libraries made with DNA inputs ranging from 1 ng to 50 ng. This integrated approach resulted in a streamlined, time-efficient, automatable workflow that yields automatically normalized libraries while maintaining high sequencing quality, minimizing duplication rates and artifact propagation. Next steps are full automation and integration of the FFPE DNA extraction from FFPE samples into the workflow, resulting in a complete solution from FFPE sample to NGS-ready, normalized library pools requiring minimal manual intervention and sample transfer.