Rapid and reliable mutational analysis of myeloid neoplasms is increasingly important for diagnostic, prognostic and therapeutic reasons. In this article we describe the development and validation of a custom next-generation sequencing (NGS) assay that reliably tests across a broad range of myeloid neoplasms, including AML, MDS, and myeloproliferative neoplasms. The lllumina TruSeq Custom Amplicon panel was designed to detect variants in 21 genes. The validation protocol included sequencing of cell lines (n=3) and patient samples (n=36) on an Illumina MiSeq platform. A read depth ≥100x was observed for >97% of targeted bases. After filtering for artifacts, a specificity of 100% was obtained. A detection limit of ≤5% was observed for variants present in cell lines. On average two reportable variants were present in samples from patients with a myeloid neoplasm. In conclusion, the custom NGS assay provides an adequate routine assay for genetic analysis of variants present in myeloid neoplasms. Practical considerations on choice of targeted genes, type of assay and method of data analysis are provided in this report.</p
