Design

RHP is based on the Illumina TSCA version 1.0 platform (Figure 1). Amplicons (approximately 250 bp) were designed with the Illumina DesignStudio and target 95 genes (Supplemental Table S1), including mutational hotspots in oncogenic driver genes and the coding sequence of tumor suppressor genes. A seven-person working group of molecular diagnosticians, hematopathologists, hematologists, oncologists, and researchers was assembled to curate from the published literature and meeting proceedings a comprehensive list of genes more frequently mutated in myeloid malignancies, acute lymphoid leukemias, and lymphoid neoplasms that commonly involve bone marrow (chronic lymphocytic leukemia, Waldenström macroglobulinemia, hairy cell leukemia, etc.). The initial list was >120 genes. The criteria used to determine whether a gene is included in the final panel were i) importance for classification according to World Health Organization; ii) risk stratification with National Comprehensive Cancer Network category 2B or above of evidence; iii) frequency of mutations; iv) germline predisposition of myeloid neoplasms; and v) primer compatibility in a multiplex panel. The amplicons (N = 1330) span 757 coding exons and 175 kb of genomic sequence in aggregate. The final design had a success rate of 98.5% and a total gap of <1% of the targeted genomic region. Approximately 50% of the target regions are covered in both directions. Difficult to design areas are mainly high-GC sequences, such as regions of CEBPA (Supplemental Table S2).

TruSeq Custom Amplicon assay workflow diagram.