The VariantPlex™ Core AML Kit was designed for superior coverage across key genes implicated in Acute Myeloid Leukemia (AML). Based on Anchored MultiPlex PCR™ (AMP) chemistry, the kit allows for fully independent, bi-directional coverage of targeted exons. Additionally molecular barcodes, or UMI’s enable unique molecule tagging for deduplication, error correction, and true coverage information for mutation detection confidence.
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For Research Use Only. Not for use in diagnostic procedures.
|FLT3||11, 13-17, 20|
|JAK2||14, 16, 19-22|
|KIT||2, 8-11, 13-15, 17, 18|
* Hotspot coverage only. For complete target information, please view protocol or panel bed file.
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 Anchored Multiplex PCR (AMP™) target enrichment chemistry to detect multiple mutation types and gene expression profiling in a single sample.
FLT3 encodes a tyrosine kinase that is frequently mutated in hematologic malignancies. Internal tandem duplications (ITDs) in FLT3 have been detected in over 20% of acute myeloid leukemia (AML) cases and confer an aggressive phenotype. Importantly, FLT3 is the target of promising new therapeutics, yet NGS-based methods often fail to detect FLT3-ITDs, as many variant callers fail to identify these highly variable repeated sequences.
The Core AML Kit contains independent and bi-directional probes specifically optimized for detecting ITDs of all sizes within the commonly mutated juxtamembrane domain and tyrosine kinase domain 1. The reads originating from those probes can be bioinformatically assembled using a de novo assembler to form longer consensus sequences that span the ITD. These longer sequences can then be aligned to the reference genome to identify the presence of an ITD.
Figure 1. GSPs (gene specific primers) in the region of FLT3 in which ITDs occur and simulated bi-directional reads that are then assembled and compared to the reference genome to detect the ITD.
To validate the VariantPlex Core AML assay and Archer Analysis FLT3-ITD detection algorithm, we tested over 20 clinical AML samples, containing seven distinct FLT3-ITDs, and over 2,000 in silico datasets representing the spectrum of known ITDs.
Figures 2 and 3. The sensitivity and specificity (for algorithm version 4) for 2000 in silico generated ITDs representing a wide range of ITDs is shown across multiple iterations of our algorithm development. The final algorithm (Version 4, launched with Archer Analysis vX.x - green) shows sensitivity of 98.2% over in silico generated ITDs and 100% specificity and sensitivity across 7 CGE-verified clinical sample types (data not shown).
ITD information is reported out in Archer Analysis, with a tiered reporting structure and event visualization in JBrowse.
Figure 4. Representative, simple visualization of an ITD event including GSP placement (red).
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. The Core AML panel is designed to have superior unique molecule coverage across all targeted genes, including challenging regions like CEPBA, DNMT3A and RUNX1.
Figure 5. Percentage of targeted bases covered at 100x unique reads. Data was compiled from 16 libraries across 3 users. 50ng gDNA input was used and samples were sequenced to a depth of 750,000 reads.
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 for. 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.
Figure 6. Average deduplicated coverage across CEBPA over 158 libraries with 50ng gDNA input. 128 oncogenic CEBPA mutations detailed in Papaemmanuil et al. NEJM 2016 are overlaid in green for hotspot reference.
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