N89: Microsatellite instability analysis and NGS with fragmented sample types

S. Peterson, S. Lewis, H. Honing, K. Oostdik, C. Knox, B. Hook

Introduction: A significant hurdle to using fragmented DNA for genomic studies is obtaining a sample of sufficient quantity and quality for rigorous downstream applications like NGS. Having effective tools to isolate, characterize, and analyze fragmented DNA containing samples, such as circulating cell free DNA (ccfDNA) and FFPE tissues, can prevent downstream failures, ultimately saving hours of work and precious samples. Here we present optimized methods for use with even highly fragmented DNA samples. Using this toolset, we demonstrate successful NGS and microsatellite instability (MSI) workflows using matched FFPE tissues and plasma samples.

Methods: Plasma and FFPE tissue samples were obtained from three individuals with colorectal adenocarcinoma. DNA was isolated with Promega’s Maxwell® RSC Instrument using the Maxwell® RSC FFPE DNA Kit for FFPE tissues and the Maxwell® RSC Circulating DNA Kit with the large volume custom protocol for plasma. DNA was then quantified with the ProNex® DNA QC Assay. Following quantitation, MSI analysis and NGS library preparation using the TruSeq Custom Amplicon Low Input Kit from Illumina was performed. NGS libraries were checked for size and quantity and then sequenced on the MiSeq® System (Illumina). Results: Full MSI profiles were obtained from DNA obtained from both ccfDNA and FFPE samples from each individual. Following successful determination of MSI-status, NGS libraries were produced from each sample. Sequencing of these libraries produced mean amplicon read depth greater than 3000x and mean coverage uniformity greater than 90%. In addition to excellent sequencing quality metrics, variants in mismatch repair genes identified in FFPE samples were also detected in matched plasma samples. Conclusions: Proper molecular tools and assays are essential to success in exacting downstream applications like NGS and multiplex PCR. This work introduces streamlined methods for DNA isolation, library preparation, and multiplex microsatellite instability analysis from fragmented sample types and demonstrates their effective use with matched FFPE and ccfDNA samples.

Top