Rebecca Biloune
Rebecca completed a PhD in Neurochemistry at UCL, and has worked as a product manager at various biotechnology companies, with focus on product portfolios including sample preparation, reagents, instrumentation, software and applications for deployment within the cell biology and NGS market. Rebecca currently works at OGT as a strategic product manager responsible for the NGS portfolio. OGT, a Sysmex Group company, is a leading global provider of clinical and diagnostic genomic solutions that are created for scientists by scientists – including CytoCell®, CytoSure® and SureSeq ranges of FISH, microarray and NGS products. OGT strives to unlock the future of genetic clinical care with a commitment to working in partnership with its customers – working closely with scientists to understand their unique challenges, and to customise its approach to meet their exact needs. Dedicated to improving clinical care, OGT believes that through partnership together we will achieve more.
Abstract
Rebecca Biloune
OGT, Begbroke, Oxfordshire, United Kingdom
Molecular technologies incorporating NGS are increasingly utilised to support traditional immunophenotypic multiparameter flow cytometry in MRD detection, including acute myeloid leukaemia (AML) disease monitoring.
We used the OGTTM Universal NGS Complete Workflow Solution library preparation kit and a focussed SureSeqTM Myeloid MRD panel to assess the applicability of a capture-based target enrichment NGS approach for studies of molecular-based MRD monitoring in AML. The panel covers 43 hotspot exons in 13 genes relevant to the AML panel (11.3 kb baited, 8 kb targeted). We used the Myeloid Reference Standard (Horizon Discovery) diluted to create frequencies in the range of 1.0 – 0.05% variant allele frequency (VAF). Sequencing was conducted using NextSeqTM (Illumina). Data was analysed using OGT’s Interpret analysis software.
Libraries captured with the Myeloid MRD panel generated from 250 ng of input DNA achieved a coverage of 20,000x when sequenced with 20 million reads per sample. We observed that increasing reads above 20 million reads marginally increased the depth and substantially increased the duplication rate and support for UMI family size filtering. We detected a 300 bp FLT3 ITD down to 0.1% and achieved 100% detection of SNVs and Indels at 0.05% in target regions, including NPM1.
Target capture NGS provides the opportunity to evaluate many genes in a single assay. Suitability for MRD requires highly uniform and sensitive target enrichment. Our study demonstrates reliable and accurate detection of variants down to 0.05% VAF, providing researchers with the capability to use capture-based NGS for Myeloid MRD monitoring.