Laura Johnson1, Marc Bessette1, Aaron Berlin1, Josh Haimes1, Darius Fugere1, Laura M. Griffin1, Katelyn Trifilo1, Namitha Manoj1, Helen Wang1, Russell Ryan2, Mohammad Hussaini3, Brian Kudlow1
1ArcherDX, Inc., Boulder, CO, USA; 2Massachusetts General Hospital, Boston, MA, USA; 3Moffitt Cancer Center, Tampa, FLRead the abstract
Hematologic malignancies can be driven by a diversity of mutation types, including single nucleotide variants, copy number variants, gene fusions, insertions and deletions and changes in gene expression profiles. However, comprehensive detection of these mutation types from a single clinical sample is challenging, as specific assays are required to detect each mutation type. Next-generation sequencing (NGS) enables comprehensive detection of all mutation types from whole genomes and transcriptomes. However, low detection sensitivity, high input requirements and high costs render these approaches impractical for routine detection of mutations from clinical sample types. Anchored Multiplex PCR (AMP™) is a target enrichment strategy for NGS that uses molecular barcoded (MBC) adapters and unidirectional gene-specific primers (GSPs) for amplification.
Our goal was to develop AMP-based NGS assays to simultaneously detect multiple mutation types from DNA and RNA, as well as relative gene expression levels and copy number alterations (CNA) relevant in hematologic malignancies. In particular, we sought to develop methods to detect novel gene fusions, internal tandem duplications (ITDs) and mutations in CEBPα.
We developed AMP-based Archer® VariantPlex™ and FusionPlex® assays to enable NGS-based detection of mutations from DNA and RNA, respectively. Open-ended amplification permits identification of novel gene fusions with FusionPlex and complex mutation types such as ITDs with VariantPlex assays. MBC adapters ligated to RNA and DNA fragments prior to amplification enable relative gene expression and CNA analysis.
We show instances of gene fusion detection from open-ended amplification in RNA, including a novel fusion, RUNX1-G6PD, in a case of acute unclassifiable leukemia. Furthermore, unidirectional GSPs provided bidirectional coverage of a BCR-ABL1 fusion, which was detected with reads originating from ABL1 as well as BCR. Using our optimized bioinformatics algorithm and the VariantPlex assay, we accurately and reliably detected ITDs of varying sizes and insertion points, with simultaneous point mutation detection, in AML-positive blood samples. Furthermore, we show multiple mutations in various AML-positive sample types, including mutations in CEBPα. Finally, MBCs used in AMP enabled NGS-based expression profiling for identification of Diffuse Large B-Cell Lymphoma subtypes in a small cohort of samples.
Our results demonstrate that AMP-based NGS enables comprehensive detection of multiple mutation types as well as gene expression levels relevant in hematologic malignancies. Importantly, AMP enables identification of known and novel gene fusions at nucleotide resolution, detection of ITDs and characterization of relative gene expression levels and CNAs.
Jens Eberlein1, Thomas Harrison1, Jennifer Sims2, Ian McKittrick1, Megan Wemmer1, Darius Fugere1, Laura M. Griffin1, Brady P. Culver1, Laura Johnson1, Brian A. Kudlow1
1ArcherDX, Inc., Boulder, CO USA; 2Q2 Solutions | EA Genomics, Morrisville NC, USARead the abstract
NGS-based analysis of the immune repertoire (IR) is a powerful tool to monitor disease, adaptive immune responses to disease, vaccination and therapeutic interventions. IR characterization by NGS usually requires large primer panels to cover its extensive combinatorial diversity, and a complex system of synthetic controls to account for differential amplification efficiency across segment combinations. Anchored Multiplex PCR (AMP™) uses molecular barcoded (MBC) adapters and gene-specific primers (GSPs), enabling NGS-based immune chain mRNA interrogation from a single side. This eliminates the need for opposing primers that bind within the highly variable V-segment, eliminating clone dropout due to somatic hypermutation.
Our goal was to develop an NGS assay based on AMP that would enable IR characterization utilizing a minimal set of unidirectional GSPs and to reduce amplification bias through the use of MBC adapters.
Upon developing our AMP-based NGS assay, we validated its quantitative reproducibility and sensitivity using mRNA isolated from PBMCs of healthy donors, B-cell chronic lymphocytic leukemia donors and formalin-fixed paraffin-embedded (FFPE) tissue.
We developed the AMP-based NGS assays, Immunoverse™ IGH and TCR, for B-cell and T-cell receptor sequencing, respectively. Both assays demonstrated high reproducibility between replicates with quantitative clone tracking down to 0.01%. The ability to determine isotype, clonotype and IGHV mutational status in a single assay was demonstrated. Preliminary TCR assay data indicates that CDR3 sequence capture is possible from FFPE tissue with clonotype calling being driven by input quantity, T-cell content, and, to a lesser degree, mRNA quality.
AMP-based NGS with MBC quantification and error-correction is a powerful method to characterize the immune repertoire.
To address the existing bottlenecks of using NGS in translational research, we’ve created a robust platform that is purpose-built for clinical oncology research.
By combining revolutionary Anchored Multiplex PCR (AMP™) chemistry with an easy-to-use workflow and intuitive software, we are unleashing the power of translational NGS to enable accurate and scalable mutation detection.
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