Abstract 1693: Comparison of commercially available myeloid next-generation sequencing assays
Authors: Sarah Johnson, Nathan Riccitelli, Reinhold Pollner
Journal: Cancer Research (2019)

Abstract Introduction Myeloid neoplasms are a complex class of disorders characterized by a range of genotypes. Accurate, precise, and rapid identification of these genotypes is crucial in giving the right drug to the right patient at the right time. In this study, we present a comparison of three Next-Generation Sequencing (NGS) panels targeting genes associated with myeloid cancers. We evaluated assay performance as a function of sensitivity and specificity of somatic variant callings, sequencing metrics, and general workflow, for the analytically validated Illumina TruSight Myeloid Sequencing Panel (TSM), the new Illumina AmpliSeq for Illumina Myeloid Panel (IA), and the ThermoFisher Oncomine™ Myeloid Research Assay (TA). Methods Thirty-two libraries were prepared across all three panels. Samples included a mix of Horizon Discovery and SeraCare myeloid DNA controls, a well-characterized Hapmap sample, and six clinical DNAs isolated from peripheral blood of patients with myeloid disorders. Libraries were prepared according to manufacturer protocols, and quantified by Qubit Flourometer. For Illumina panels, libraries were manually prepared, normalized, pooled, and sequenced on Illumina’s MiSeq instruments. For the TA panel, libraries were generated, pooled, templated, and loaded onto sequencing chips by the IonChef, then sequenced on the S5xL system. Primary data analysis was completed using cloud-based bioinformatic solutions followed by manual annotation. Results Hands-on time varied for the assays, with the TA panel requiring just 30 minutes compared to four to eight hours for the TSM and IA protocols. Time to results from DNA dilution to variant calls was comparable, at three days for the TSM panel and four days for the IA and TA protocols. The TSM assay exhibited the highest average coverage, but also the greatest amplicon dropout. Both the IA and TA panels exhibited similar coverage uniformity and read depths. Detection of expected variants in control samples down to 5% allele frequency was robust for all assays, however both the IA and TA panels contained increased regions of poor sequencing quality relative to the TSM panel. No false positives were found in the somatic-variant negative Hapmap sample NA12878, and concordance of variant calls for the clinical samples from the IA and TA assays to the validated TSM assay was greater than 95%. Conclusions In the complex landscape of myeloid neoplasms, NGS is an invaluable tool for the diagnosis and management of the diseases. Overall, the three sequencing panels evaluated herein provided accurate genotype information for actionable targets in myeloid cancers. Although results for the assays were similar, the short time-to-data for the TSM assay, increased coverage and detection of low frequency variants for the IA assay, and fully-automated option for the TA assay should all be considered when determining the optimal panel for a given need. Citation Format: Sarah Johnson, Nathan Riccitelli, Reinhold Pollner. Comparison of commercially available myeloid next-generation sequencing assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1693.