This assay detects single nucleotide variants (SNVs), short insertion-deletions and selected gene fusions in 164 genes recurrently altered in myeloid and lymphoid neoplasms. The Stanford Actionable Mutation Panel for Hematopoietic and Lymphoid Malignancies (Heme-STAMP) is a targeted next generation sequencing method. The workflow includes acoustic shearing of isolated genomic DNA, followed by efficient preparation of sequencing libraries and a targeted enrichment approach to capture genomic regions of interest. The enrichment is accomplished using custom designed libraries of capture oligonucleotides that target a specific set of genomic regions. This panel targets 164 genes, either in part or fully, with the genes selected on the basis of their known impact as actionable targets of existing and emerging anti-cancer therapies, their prognostic features, and/or their mutation recurrence frequency across patients with hematopoietic neoplasms. These genomic features are interrogated to achieve a minimum analytic detection-limit of 5% for SNVs and insertion-deletion variants. Pooled libraries are sequenced on an Illumina sequencing instrument.

Due to inherent limitations of the NGS method, insertion-deletion variants larger than 25 bp are not reliably detected. To detect larger insertion and deletions in key regions of CALR and FLT3, PCR amplification of these regions is performed, followed by capillary electrophoresis fragment analysis.