||This test, based on the TruSight© Myeloid Sequencing Panel (Illumina©), is an amplicon-based next generation sequencing (NGS) assay designed to detect single nucleotide variants (SNVs) and insertion-deletion variants in 54 genes that are recurrently mutated in myeloid neoplasms, including acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPNs), chronic myelomonocytic leukemia (CMML), and juvenile myelomonocytic leukemia (JMML). The method begins with hybridization of optimized oligonucleotide probes upstream and downstream of the regions of interest. Each probe includes a target capture sequence and an adapter sequence used in subsequent amplification reaction. A proprietary extension-ligation reaction (Illumina©) extends across the region of interest, followed by ligation to unite the two probes. This creates a new template strand and gives the assay excellent specificity. Extension-ligation templates are PCR amplified and two unique sample specific indices are incorporated. The final reaction product contains amplicons that are ready for sequencing. An integrated bead-based normalization procedure allows for volumetric library pooling. Pooled amplicon libraries are loaded directly onto the MiSeq system.
PCR amplification followed by Sanger sequencing is performed to detect variants in CEBPA, which is poorly covered by the NGS method.
Due to inherent limitations of the NGS method, insertion-deletion variants larger than 20 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.
This test is not intended to diagnose inherited diseases, and reported variants are only annotated for their relevance to cancer. If there is clinical suspicion for a non-cancer related inherited disease involving a gene on this panel, separate testing of a germline specimen is suggested.
Genes tested by Sanger sequencing: CEBPA
Genes tested by fragment size analysis: CALR, FLT3
Genes tested by Next Generation Sequencing:
For information on specific regions/exons tested, please contact the Molecular Pathology Lab at 650-723-6574.