Liquid biopsies have the potential to be a less invasive method than traditional biopsies to detect advanced solid tumor mutational status. Anchored Multiplex PCR (AMP™) is target enrichment chemistry for NGS that is uniquely suited for highly fragmented material such as liquid biopsy-derived circulating tumor DNA (ctDNA). AMP-based ctDNA library preparation uses molecular barcoded adapters to remove PCR duplicates, correcting for both PCR and sequencer-derived errors while enabling accurate allele frequency quantitation.
Josh Stahl, CSO and general manager at ArcherDX is originally from Baltimore and studied biochemistry and molecular biology at West Virginia University. He went to graduate school at the University of Colorado, Boulder, working on protein-protein interactions in morphine analgesia.
Josh joined ArcherDX in 2013 and has overseen biochemistry,molecular biology, and software development. Now general manager and CSO, Josh continues to lead Archer R&D, product development and operations teams in developing new applications and Archer technology.
Anchored Multiplex PCR Enables Sensitive and Specific Detection of Variants in Circulating Tumor DNA by Next-Generation Sequencing
Jerome E. Lee1, Namitha Manoj1, Josh D. Haimes1, Skyler J. Mishkin1, Paula G. Roberts1, Eric M. Davis1, Ian McKittrick1, Sandra Elmore2, Laura M. Griffin1, Ryan D. Walters1, Brian A. Kudlow1, Margaret L. Gulley2, Brady P. Culver1
1 ArcherDX, Boulder, CO USA
2 University of North Carolina School of Medicine, Chapel Hill, NC
Introduction: Liquid biopsies are a promising, minimally invasive alternative to tissue biopsies that have potential cost, time and safety benefits, as well as a greater ability to interrogate heterogeneous tumors. However, except in advanced disease states, cell free DNA (cfDNA) is typically of low abundance and only a small portion of cfDNA originates from tumor cells as circulating tumor DNA (ctDNA), which tends to be highly fragmented (100-300bp). Therefore, NGS-based assays to detect variants in ctDNA must be sensitive enough to detect mutations at allele frequencies (AF) <2% from <100ng of highly fragmented DNA.
Methods: We developed the Archer® Reveal ctDNA™ 28 assay based on Anchored Multiplex PCR (AMP™), a target enrichment method for NGS that uses unidirectional gene-specific primers and molecular barcoded (MBC) adapters for amplification. AMP is well suited to amplify small cfDNA fragments, as it only requires one intact primer-binding site within a fragment. Single primers capture target regions from both strands independently, increasing the sensitivity of variant detection from low-input samples. MBC adapters ligated prior to amplification permit post-sequencing error correction, reducing background noise and increasing analytical sensitivity of ultra low-allele frequency variant detection. Finally, variant filtering in the Archer Analysis pipeline further increases the specificity of variant calls.
Results: Using commercially available reference ctDNA standards, we demonstrate that genomic DNA present in plasma does not significantly impact amplification of small, fragmented ctDNA with the AMP-based Reveal ctDNA 28 assay. Based on sequenced reads, AMP enabled interrogation of more than 65% of the input molecules from 50ng starting material. As a result, we show 100% detection sensitivity for 1% AF variants using 10ng DNA input and 71.9% detection sensitivity for 0.1% AF variants using 50ng DNA input. MBC-enabled post-sequencing error correction and variant filtering reduced the number of false positives by 98%, resulting in 91.7% specificity. Finally, mutations detected from liquid biopsy-derived ctDNA showed cancer type-dependent concordance with tissue biopsy findings, and revealed additional oncogenic driver mutations.
Conclusions: The Archer Reveal ctDNA 28 assay is a powerful tool for sensitive and specific NGS-based detection of variants in ctDNA, demonstrating accurate allele frequency quantification of synthetic reference standards. This assay is a promising approach to characterize solid tumors from liquid biopsies, showing cancer type-dependent concordance of tissue and plasma mutation profiles, as well as identification of additional oncogenic driver mutations in ctDNA.
Characterization of hematologic malignancies with Anchored Multiplex PCR and next-generation sequencing
Laura Johnson1, Marc Bessette1, Aaron Berlin1, Josh Haimes1, Laura M. Griffin1, Katelyn Trifilo1, Namitha Manoj1, Helen Wang1, Russell Ryan2, Mohammad Hussaini3, Brian Kudlow1
1 ArcherDX, Inc., Boulder, CO, USA
2 Massachusetts General Hospital, Boston, MA, USA
3 Moffitt Cancer Center, Tampa, FL
Introduction: 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. We developed targeted next-generation sequencing (NGS) assays based on Anchored Multiplex PCR (AMP™) for simultaneous detection of mutations and gene expression levels relevant in hematologic malignancies.
Methods: AMP is a library preparation method for NGS that uses molecular barcoded (MBC) adapters and unidirectional gene-specific primers (GSPs) for amplification. AMP-based Archer® VariantPlex™ and FusionPlex® assays 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 internal tandem duplications (ITDs) with VariantPlex assays. MBC adapters ligated to RNA fragments prior to amplification enable relative gene expression analysis.
Results: We show that open-ended amplification from KMT2A GSPs enabled detection of a KMT2A-MLLT3 fusion through breakpoint identification, with reads extending 6 exons into MLLT3. We also detected a novel RUNX1 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. 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.
Conclusions: 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.
Characterization of B- and T-cell Immune Repertoires Using Anchored Multiplex PCR and Next-Generation Sequencing
Jens Eberlein1, Thomas Harrison1, Jennifer Sims2, Ian McKittrick1, Megan Wemmer1, Laura M. Griffin1, Brady P. Culver1, Laura Johnson1, Brian A. Kudlow1
1 ArcherDX, Inc., Boulder, CO USA
2 Q2 Solutions | EA Genomics, Morrisville NC, USA
Introduction: 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. Here, we describe AMP-based NGS assays for IR characterization, Immunoverse™ IGH and TCR, which utilize a minimal set of unidirectional GSPs and MBC adapters that reduce amplification bias.
Methods: The quantitative reproducibility and sensitivity of our assays was validated using mRNA isolated from PBMCs of healthy donors, B-cell chronic lymphocytic leukemia donors and formalin-fixed paraffin-embedded (FFPE) tissue.
Results: 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.
Conclusions: AMP-based NGS with MBC quantification and error-correction is a powerful method to characterize the immune repertoire.
The Archer® VariantPlex® CFTR kit is a targeted NGS assay for comprehensive detection of known and unknown variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
ArcherDX addresses the bottlenecks associated with using NGS by offering a robust platform for targeted sequencing applications.Archer target enrichment assays utilize AMP chemistry to generate highly enriched sequencing libraries for comprehensive profiling of fusions, CNVs, SNVs and indels. Amplification from independent, unidirectional primers and universal molecular barcoded adapters permit identification of novel gene fusions and mutations with nucleotide-level resolution. Requiring only one intact primer-binding site, AMP chemistry is uniquely suited to amplify small, degraded fragments, enabling solid tumor mutation profiling from FFPE samples and liquid biopsies. Archer’s easy-to-use, lyophilized kits generate sequencing-ready libraries from RNA, DNA, and liquid biopsy-derived ctDNA. Complemented by the Archer suite of assay design and bioinformatics analysis, Archer’s FusionPlex, VariantPlex and Reveal ctDNA assays facilitate complex mutation identification and discovery, while Immunoverse assays enable quantitative profiling of the expressed immune repertoire.
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