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
Introduction: Deregulation of the proto-oncogene, MET, confers an aggressive phenotype in a variety of human cancers, promoting proliferation, invasive growth and angiogenesis. MET deregulation can be driven by gene amplification, overexpression, exon 14 skipping, gene fusions and single nucleotide variants (SNVs), such as kinase-activating point mutations. MET is a target of intensive drug development efforts, although the various mutated forms of MET exhibit unique drug sensitivities. Therefore, detection of these mutations has an important role in the development of drugs targeting MET, and has the potential to guide treatments for cancers driven by MET deregulation. We developed a targeted NGS assay based on Anchored Multiplex PCR (AMP™) to detect all types of mutations driving MET deregulation from a single sample.
Methods: AMP only requires a single gene-specific primer for amplification, enabling open-ended capture of DNA and cDNA fragments for NGS-based detection of known and unknown mutations. We developed AMP-based Archer? VariantPlex™ and FusionPlex? library preparation assays to detect mutations from DNA and RNA, respectively. AMP probes were designed to cover the MET gene for detection of copy numbers variants (CNVs) and SNVs from DNA, and known and novel fusions, exon skipping and expression levels from RNA.
Results: We show that the VariantPlex assay enables NGS-based detection of MET amplifications from DNA in concordance with FISH results. Further NGS analysis of RNA from the same sample using the FusionPlex assay revealed the resulting overexpression of MET. We also demonstrate that AMP-enabled open-ended capture of cDNA fragments allows for reliable detection of exon 14 skipping in FFPE samples and in cells, consistent with RT-PCR results. Parallel analysis of DNA from the cell samples revealed splice site mutations that have been previously reported to drive exon 14 skipping. Furthermore, this open-ended capture also permitted identification of a novel GTF2I:MET gene fusion in a patient-derived xenograft model. Finally, we detected an kinase-activating point mutation in MET, p.Y1253D, by analysis of genomic DNA with the VariantPlex NGS assay.
Conclusions: These results show that AMP-based VariantPlex and FusionPlex Assays enable comprehensive detection of multiple mutation types from low-input clinical sample types, such as FFPE specimens. As MET deregulation can be driven by many different genetic aberrations, this allows for NGS-based characterization of MET deregulation from a single sample.
Rapid and Comprehensive Detection of CFTR Variants Across Ethnic Groups using Anchored Multiplex PCR and Next-Generation Sequencing
Matthew T. Hardison, PhD, FACMG, Laura M. Griffin, PhD, Brady P. Culver, PhD
Introduction: Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. CF is characterized by the build-up of thick mucus resulting in chronic lung infections and airway inflammation. Carrier identification and newborn screening have significant implications in the overall prognosis of CF patients. Underlying CFTR mutations were recently shown to vary significantly across ethnic groups. However, current CFTR genotyping assays detect mutations highly prevalent in white individuals, yet fail to detect mutations that are more prevalent in nonwhite individuals. We present a rapid, cost-effective assay for comprehensive detection of CFTR mutations for pan-ethnic carrier identification and newborn screening.
Methods: BabyGenes, Inc, in partnership with ArcherDX, Inc., has developed a targeted next-generation sequencing (NGS) assay based on Anchored Multiplex PCR (AMP™) to detect mutations in 105 genes clinically linked to inborn errors in metabolism, including CFTR. AMP is a library preparation method for NGS that uses unidirectional gene-specific primers (GSPs) and molecular barcoded adaptors ligated to random start sites to enrich for both known and unknown mutations across a panel of target regions. Following analytical and clinical validation of the panel, a total of 1,585 clinical samples representing a diverse ethnic set were analyzed for clinically significant CFTR variants.
Results: Clinical validation with 150 blinded specimens from the Coriell Institute for Medical Research resulted in 100% accuracy of variant detection within the CFTR gene. Pan-ethnic screening of 1,585 clinical samples identified 34 unique mutations, several of which were identified in multiple individuals. 73% (25/34) of these unique mutations and 60% (74/123) of total mutations detected are not currently included in the ACMG-recommended 23-mutation panel for CF carrier screening. Furthermore, this screening revealed ethnic differences in clinically significant CFTR variants and a pan-ethnic carrier rate of approximately 7%.
Conclusions: We demonstrate that the BabyGenes, Inc. AMP-based targeted NGS assay enables rapid, highly sensitive, and comprehensive detection of both known and novel mutations in the CFTR gene. This is critical for global carrier and newborn screening, as CF driver mutations have not been fully characterized across all ethnicities. Findings suggest that the pan-ethnic carrier rate of CF may be higher than originally predicted. As this entire assay can be performed in under 96 hours and the reagents do not require refrigeration, AMP is a practical and economical method for global communities.
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
1ArcherDX, Boulder, CO, USA
2University of North Carolina School of Medicine, Chapel Hill, NC, USA
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
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
1 ArcherDX, Inc., Boulder, CO, USA
2Massachusetts General Hospital, Boston, MA, USA
3Moffitt Cancer Center, Tampa, FL, USA
Introduction: Hematologic malignancies can be driven by a diversity of mutation types, including single nucleotide variants (SNVs), copy number variants (CNVs), gene fusions, insertions and deletions (indels) 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™) to simultaneously detect multiple mutation types, including but not limited to novel gene fusions and ITDs, as well as relative gene expression levels relevant in hematologic malignancies.
Methods: AMP is a library preparation method for NGS that uses molecular barcoded (MBC) adapters and single gene-specific primers (GSPs) for amplification. We developed AMP-based Archer? VariantPlex™ and FusionPlex? assays for NGS-based detection of mutations from DNA and RNA, respectively. Open-ended amplification permits identification of both known and novel gene fusions with FusionPlex assays. Furthermore, we developed a VariantPlex assay and novel bioinformatics algorithm to detect ITDs from clinical DNA samples. Finally, MBC adapters ligated to RNA fragments prior to amplification enable determination of relative gene expression levels.
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. AMP also enabled NGS-based detection of a novel RUNX1 fusion, RUNX1-G6PD, in a case of acute unclassifiable leukemia. Furthermore, we show that single, unidirectional GSPs provide bidirectional coverage of a BCR-ABL1 fusion, which was detected with reads originating from ABL1 as well as BCR GSPs. 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, we show NGS-based expression profile analysis with the FusionPlex assay, resulting in 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.
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
Introduction: The adaptive immune system is involved in various disease conditions including cancer, chronic infection, autoimmune disease and transplant rejection. Adaptive immunity is mediated by B and T lymphocytes, which are activated upon antigen binding to antigen receptors expressed on their surface. Therefore, the spectrum of these antigen receptors, or immune repertoire (IR), provides a means to monitor adaptive immune responses to disease, vaccination and therapeutic interventions. Next-generation sequencing (NGS) of antigen receptor genes is a valuable tool in the study of disease states and responses to various interventions. Traditional amplicon-based NGS assays use opposing primers for targeted amplification of rearranged antigen receptor genes. Thus, large primer panels are required to capture the extensive combinatorial diversity exhibited by the IR. Quantification from such assays requires a complex system of synthetic controls to account for differential amplification efficiency across segment combinations. Here, we describe an Anchored Multiplex PCR (AMP)-based NGS assay to analyze the IR, employing a minimal set of gene-specific primers in conjunction with molecular barcodes (MBCs) to reduce amplification bias.
Methods: AMP uses MBCs ligated to cDNA ends and gene-specific primers for amplification, enabling 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. Furthermore, this facilitates CDR3 sequence capture from highly fragmented RNA inputs.
Results: We validated the quantitative reproducibility and sensitivity of the AMP-based IGH assay using mRNA isolated from peripheral blood leukocytes of healthy and B-cell chronic lymphocytic leukemia (B-CLL) donors. Our data showed high reproducibility between replicates and quantitative clone tracking down to 0.01%, with the ability to determine IGHV mutational status. We also validated the quantitative reproducibility and sensitivity of the AMP-based T-cell receptor (TCR) assay using high-quality mRNA isolated from peripheral blood leukocytes and highly fragmented RNA isolated from formalin-fixed paraffin-embedded (FFPE) samples. Our data indicate that clonal diversity in sequencing data is driven by input quantity, total T-cell number, 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.
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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