Brian A. Kudlow1, Josh Haimes1, Marc Bessette1, Namitha Manoj1, Laura M. Griffin1, Danielle Murphy2, Robert Shoemaker2, Joshua Stahl1.
1ArcherDX, Boulder, CO, USA 2Ignyta, San Diego, CA, USA
Deregulation of the proto-oncogene, MET, confers an aggressive phenotype in a variety of human cancers and can be driven by gene amplification, overexpression, exon 14 skipping, gene fusions and kinase-activating point mutations. MET is a target of intensive drug development efforts, although the various mutated forms of MET exhibit unique drug sensitivities. Here, we describe a targeted NGS assay based on Anchored Multiplex PCR (AMP™) to detect all types of mutations driving MET deregulation from a single sample. AMP only requires a single gene-specific primer for amplification, enabling open-ended capture of DNA and cDNA fragments. Using this assay, we detected MET amplifications and resulting overexpression, exon 14 skipping with concomitant splice site mutations, a novel GTF2I:MET fusion and a Y1253D activating point mutation. Many of these results were obtained from low-input FFPE specimens, demonstrating that AMP-based NGS enables comprehensive detection of all types of MET mutations from clinical samples.
Pan-Ethnic Detection of CFTR Variants using Anchored Multiplex PCR and Next-Generation Sequencing
Matthew T. Hardison, PhD, FACMG, Laura M. Griffin, PhD, Brady P. Culver, PhD
Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene. Underlying CFTR mutations were recently shown to vary significantly across ethnic groups. We present a rapid, cost-effective NGS assay based on Anchored Multiplex PCR (AMP™) for comprehensive detection of CFTR mutations across ethnic groups. AMP uses unidirectional gene-specific primers and molecular barcoded adapters for amplification of known and unknown mutations. We show 100% accuracy of CFTR variant detection, and identified 34 unique mutations in a blinded screen of 1,585 U.S. clinical samples. This revealed ethnic-specific CFTRvariants, 73% of which are not included in current ACMG guidelines for CF carrier screening. Furthermore, we revealed a pan-ethnic carrier rate of ~7%, higher than previously predicted. Therefore, global CF screening will require pan-ethnic identification of CFTR variants. Our AMP-based NGS assay is practical for global communities, as it can be performed in under 96 hours with all lyophilized reagents.
Sensitive and Specific Variant Detection in Circulating Tumor DNA by Anchored Multiplex PCR and NGS
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
1ArcherDX, Boulder, CO, USA
2University of North Carolina School of Medicine, Chapel Hill, NC, USA
Liquid biopsies are a promising, minimally invasive alternative to tissue biopsies with potential cost, time and safety benefits, as well as a greater ability to interrogate heterogeneous tumors. We developed the Archer® Reveal ctDNA™ 28 assay based on Anchored Multiplex PCR (AMP™) for NGS-based detection of mutations in circulating tumor DNA (ctDNA) derived from liquid biopsies. This assay demonstrates 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. We show that molecular barcodes used in AMP enable post-sequencing error correction, 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. These results indicate that the Archer Reveal ctDNA 28 assay is a powerful tool for sensitive and specific NGS-based detection of variants in liquid biopsies.
Hematologic Malignancy Characterization by Anchored Multiplex PCR and Next-Generation Sequencing
Josh Haimes1, Laura Johnson1, Benjamin Van Deusen1, Marc Bessette1, Aaron Berlin1, Namitha Manoj1, Aaron Berlin1, Michael Banos1, Erik Reckase1, Laura M. Griffin1, Katelyn Trifilo1, Abel Licon1, Helen Wang1, Skyler Mishkin1, Thomas Harrison1, Russell Ryan2, Mohammad Hussaini3, Joshua Stahl1, Brian Kudlow1
1ArcherDX, Inc., Boulder, CO, USA
2Massachusetts General Hospital, Boston, MA, USA
3Moffitt Cancer Center, Tampa, FL, USA
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. We developed targeted NGS assays based on Anchored Multiplex PCR (AMP™) to simultaneously detect multiple mutation types. AMP is a library preparation method for NGS that uses molecular barcoded (MBC) adapters and single gene-specific primers for amplification. We demonstrate that AMP-based NGS identifies novel gene fusions relevant in blood cancers from a single end through breakpoint identification. We also show that strand-specific amplification permits bidirectional coverage for internal confirmation of fusion events. Furthermore, we optimized our bioinformatics algorithm to accurately detect ITDs along with concomitant point mutations in AML 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.
B- and T-Cell Immune Repertoire Characterization by Anchored Multiplex PCR and Next-Generation Sequencing
Jens Eberlein, Thomas Harrison, Ian McKittrick, Megan Wemmer, Laura M. Griffin, Brady P. Culver, Laura Johnson, Brian A. Kudlow
The immune repertoire (IR) provides a means to monitor adaptive immune responses to disease, vaccination and therapeutic interventions. NGS-based IR characterization usually requires large primer panels to capture its extensive combinatorial diversity and a complex system of synthetic controls to account for differential amplification efficiency across segment combinations. Anchored Multiplex PCR (AMP™) is a library preparation method for NGS that uses molecular barcoded (MBC) adapters and gene-specific primers for amplification, enabling immune chain mRNA interrogation from a single side. We developed an AMP-based NGS assay for IR characterization with a minimal set of unidirectional gene-specific primers and MBCs that reduce amplification bias. We show high reproducibility between replicates and quantitative clone tracking down to 0.01%, with the ability to determine IGHV mutational status. Our data indicate that clonal diversity in sequencing data is primarily driven by input quantity and total T-cell number.
Mutation profiling from circulating tumor DNA in liquid biopsies
The Archer® Reveal ctDNA™ 28 Kit for Illumina® is an advanced and user-friendly solution for targeted NGS of circulating cell-free tumor DNA e.g., ctDNA, ccfDNA, cfDNA from 28 genes commonly found mutated in solid tumor type cancers.
Archer® Immunoverse™ kits are targeted NGS assays to characterize the human immune repertoire from RNA input. Powered by AMP, the lyophilized kits uniquely tag and amplify V(D)J rearrangements for sequencing on Illumina® platforms.
Comprehensive mutation profiling for cystic fibrosis
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.
For Research Use Only. Not for use in diagnostic procedures.
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