Archer® Blood Cancer assays detect various driver mutation types in myeloid and lymphoid malignancies by targeted next-generation sequencing (NGS). This approach combines FusionPlex® and VariantPlex® kits to characterize gene fusions, CNVs and other variants from a single sample.
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AML mutation detection using error-corrected sequencing
FLT3-ITD detection using Archer Blood Cancer NGS assaysg
See the FLT3-ITD AMP data here
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 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. 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.
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 superior 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. 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 bi-directional coverage of target exons, including traditionally difficult regions like CEBPA.
Reads originating from unidirectional gene-specific primers (GSPs) result in open-ended capture of gene fusions. For clinically relevant known fusion genes, GSPs are designed to capture the fusion event from both ends, enabling independent detection of a single fusion event in a single assay, thus providing internal, orthogonal verification of these important fusions. In the adjacent example, 409 unique reads originating from ABL1 GSPs and 329 unique reads originating from BCR independently detected a BCR-ABL1 fusion. Furthermore, digital read counting of molecular barcodes (MBCs) ligated prior to amplification can be used to assess expression changes across the fusion gene to detect expression imbalance, providing a third internal confirmation of the detected fusion.
Molecular barcodes ligated to RNA fragments prior to amplification enable determination of relative abundance of unique RNA fragments. This information can be used to assess relative expression levels of select critical genes, such as CD274 (PD-L1) expression in FFPE samples shown in the figure (left part of figure). Gene expression profiles can also be used to identify the cellular origins of tumor cells (COO) and for cancer subtype stratification, such as the classification of diffuse large B-cell lymphoma (DLBCL) subtypes shown above (right part of figure).
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