Introduction Acute myeloid leukemia (AML) oncogenesis is thought to require multiple somatic mutations in a "two-hit" process to 1) increase proliferation and 2) prevent maturation of myeloid cells. While FLT3 and KIT mutations are associated with increased proliferation, NPM1, CEBPA and several other mutations can be associated with maturation inhibition. The most common mutations in AML are internal tandem duplications (ITDs) in FLT3, which are detected in more than 20% of pediatric and adult AML cases and are associated with an aggressive phenotype. As FLT3-ITD expressed kinases are sensitive to tyrosine kinase inhibitors, they are of considerable interest for the development of novel AML treatments. Capillary gel electrophoresis can detect ITDs but cannot be easily coupled with assays to detect other mutation types common in AML. Next-generation sequencing (NGS)-based methods enable comprehensive detection of multiple mutation types. However, detection of ITDs by NGS is particularly challenging, in part because of their highly variable nature and the difficulties of mapping repeated sequences to a wild-type reference. Anchored Multiplex PCR (AMP) is a target enrichment strategy for NGS that uses molecular barcoded adaptors and gene-specific primers, permitting open-ended capture of DNA fragments from a single end. We present an approach based on AMP technology and a novel bioinformatics algorithm to detect ITDs in FLT3 as well as other mutation types common in AML. Methods We developed a library preparation assay for NGS to detect FLT3-ITDs as well as other mutations relevant in AML from genomic DNA extracted from clinical samples. We designed AMP probes to cover the commonly mutated juxtamembrane domain and tyrosine kinase domain 1. We further developed a novel de novo sequence assembly algorithm based on over 2000 in silico datasets representing a large range of known ITDs. Results In silico datasets enabled optimization of our sequencing data analysis algorithm, resulting in the detection of over 98% of in silico ITDs with no false positives. The AMP library preparation assay in conjunction with the optimized analysis algorithm enabled sensitive NGS-based detection of ITDs in 16 AML-positive blood samples. These results were consistent with results obtained from standard capillary gel electrophoresis. In addition, point mutations in the TKD of FLT3 and insertions in NPM1 exon 11 were detected in 2/7 and 5/7 FLT3-ITD positive blood samples. Conclusions Our data show that AMP enables accurate NGS-based detection of FLT3-ITDs from clinical DNA samples. As this approach can detect multiple mutation types from a single sample, our AMP library preparation coupled with our NGS analysis algorithm enables simultaneous detection of multiple mutations relevant in AML. Disclosures Van Deusen:ArcherDX, Inc.: Employment. Bessette:ArcherDX, Inc.: Employment. Johnson:ArcherDX, Inc.: Employment. Berlin:ArcherDX, Inc.: Employment. Banos:ArcherDX, Inc.: Employment. Griffin:ArcherDX, Inc.: Employment. Reckase:ArcherDX, Inc.: Employment. Stahl:ArcherDX, Inc.: Employment. Licon:ArcherDX, Inc.: Employment. Kudlow:ArcherDX, Inc.: Employment.