Open-ended amplification with defined read structure allows for de novo assembly and robust ITD detection of all sizes and integration sites.
Internal tandem duplications (ITDs) in FLT3 are common oncogenic drivers in acute myeloid leukemia (AML) which often coexist with other types of driver mutations. Although NGS simultaneously detects multiple mutation types in a single sample, ITDs pose unique challenges to NGS methods, in part because of their highly variable nature and the difficulties of mapping repeated sequences to a wild-type reference. Anchored Multiplex PCR (AMP) technology permits complete, bidirectional coverage of ITD-containing regions, and the Archer Analysis pipeline enables de novo assembly of sequencing reads to generate a consensus sequence. As shown in the figure above, AMP-based NGS in combination with Archer Analysis enabled FLT3-ITD detection from blood samples in concordance with capillary gel electrophoresis (CGE), the current gold standard method for ITD detection.
High-complexity coverage across CEBPA
De-duplication of reads allows for true coverage information – i.e. the reported coverage depth represents the number of unique input molecules that were captured and sequenced, not the number of PCR duplicates that were sequenced. VariantPlex Myeloid panels are designed to have maximum unique molecule coverage across all targeted genes, including challenging regions like CEPBA, DNMT3A and RUNX1.
CEBPA contains 75% GC content over its coding region, making amplification and sequencing of the region challenging. Anchored Multiplex PCR (AMP) technology is well suited for amplification of this region due to flexible primer design strategies. Unlike opposing primer–based techniques, only one primer location needs to be designed. These primers are independent and can be moved around to give the best chance of amplification across the gene. Additionally, since Archer libraries and mutation calls are based on unique reads rather than PCR duplicates, variant calling can be made with library-specific knowledge of sensitivity.
Complete, strand-specific and bidirectional coverage of target exons,
including traditionally difficult regions like CEBPA.