Comprehensive mutational profiling in blood cancers is increasingly important, yet there are challenges with existing NGS-based approaches. Complex mutation types like gene fusions and FLT3-ITDs are difficult for standard chemistries and bioinformatic approaches. Critical genomic regions like CEBPA are notoriously difficult to detect variants with confidence. Archer's proprietary AMP™ chemistry and purpose-built bioinformatic suite — Archer Analysis — address these critical concerns.
From easy-to-use lyophilized reagents, Archer NGS assays generate highly enriched sequencing libraries to detect gene fusions, point mutations, indels, CNVs and RNA abundance. Post-sequencing, Archer Analysis incorporates state-of-the-art error correction and noise characterization for confident mutation detection and reporting from a simple user interface.
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AMP chemistry is flexible and gene-level probe sets are modular. This means that any of our 94 wet lab-validated designs can be added to any preconfigured panel to build assays that fit your research goals. Archer Assay Designer gives you the power to easily determine assay content and keep current with the latest research.
Best-in-class detection of known and novel gene fusions with integrated Quiver™ fusion database
Precise breakpoint identification of splicing events like MLL, PTD and IKZF1
Confidently detect SNVs with powerful sequencing noise characterization and molecular barcodes, even in tough regions like CEBPα
Differentiate lymphoma subtypes with molecular barcode-enabled single-gene expression level in critical genes like PDL1 and multi-gene expression signatures
AMP technology and purpose-built analysis enable robust detection of complex mutations like the large CALR deletion and the widely variable and important FLT3-ITDs
Unique molecule counting enables sensitive CNV detection at single-gene level or chromosomal arm deletion/gain inference
Gene fusions are frequently observed in hematologic malignancies. For example, 302 fusions have been identified in AML and 237 fusions have been identified in ALL, and in both cases over 50 of these fusions are recurrent (F. Mertens et al, 2015). Furthermore, some genes are highly promiscuous, forming fusions with multiple different gene partners. An example of this is shown adjacently, where greater than 66 fusion partners have been discovered for MLL (or KMT2A), a frequently rearranged gene in AML. While break-apart FISH (below) can detect both known and novel fusions, it lacks the scalability to detect multiple fusions in a single sample. NGS-based methods enable highly multiplexed interrogation of gene fusions, however opposing-primer based techniques (bottom right) that are used to enrich for target regions of interest require prior knowledge of both fusion partners, thus limiting their ability to detect novel fusions. As new fusions are continually discovered, designing new primers to detect each new fusion partner is impractical.
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.
The molecular landscape of leukemia and lymphoma has expanded exponentially in the last two decades, and the complexity and scope of biomarkers makes molecular analysis difficult for any single approach. Archer Blood Cancer assays are powered by AMP target enrichment chemistry to detect multiple mutation types and gene expression profiling in a single sample.
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).
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.
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