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Detection of copy number variants by next generation sequencing is driven by genomic DNA integrity

Josh D. Haimes, James Covino, Namitha Manoj, Elina Baravik, Laura Johnson, Laura M. Griffin, Joshua Stahl, Brady P. Culver, Brian Kudlow

ArcherDX, Inc., Boulder, CO

Copy number variants (CNVs) are common oncogenic drivers, impacting more of the cancer genome than all other types of mutations combined. Next generation sequencing (NGS) of cancer genomes is a highly sensitive method to detect CNVs from clinical sample types. However, as FFPE storage of clinical specimens severely damages DNA and results in poor sequencing coverage, detection of low-level CNVs (<3-fold) and CNVs in samples with low tumor cellularity remains challenging.

Anchored Multiplex PCR (AMP™) is a target enrichment strategy that increases the read depth of target sequences by NGS. Molecular barcoded adapters ligated to DNA fragments prior to PCR enables amplification of fragments as small as 50bp. AMP therefore increases read depth and coverage of target regions, thus enhancing the detection sensitivity of CNVs by downstream NGS assays.

Since DNA damage from FFPE storage limits the number of molecules available for library preparation, the amount of amplifiable DNA fragments, rather than the total mass of DNA, drives library complexity, sequencing coverage, and detection sensitivity. To demonstrate the effect of input genomic DNA integrity on CNV detection sensitivity, we used the Archer PreSeq™ DNA QC Assay to determine the amounts of amplifiable genomes from over 150 FFPE tumor samples. AMP-based NGS using Archer VariantPlex assays in conjunction with Archer Analysis detected CNVs down to 2-fold and demonstrated that input genomic DNA integrity predicts the detection limit of CNVs. The accuracy of low-level CNV detection was also confirmed by serial dilution of CNV-positive samples.

Anchored Multiplex PCR enables comprehensive profiling of thyroid and lung cancer mutations by next generation sequencing

Josh Haimes, Laura Johnson, James Covino, Namitha Manoj, Marc Bessette, Elina Baravik, Abel Licon, Ryan D. Walters, Laura M. Griffin, Brady P. Culver, Joshua A. Stahl, Brian A. Kudlow

ArcherDX, Inc., Boulder, CO

Thyroid and lung cancers may be driven by genetic aberrations, including single nucleotide variants (SNVs), insertions and deletions (indels), copy number variants (CNVs) and fusions. Thyroid cancer subtypes require different treatment modalities and can be distinguished based on their unique driver mutations. Driver mutations in non-small cell lung cancers (NSCLCs) are often targetable with highly specific therapies. It is therefore necessary to simultaneously detect multiple mutation types in multiple genes from a single sample.

Next generation sequencing (NGS) of target-enriched samples is a highly sensitive and scalable methods to detect these mutations. Anchored Multiplex PCR (AMP™) is a target enrichment strategy for NGS that uses unidirectional gene-specific primers and molecular barcode adapters ligated to fragmented ends of genomic DNA or cDNA for amplification. This enables amplification of both known and unknown mutations within target regions and increases coverage of target regions.

We developed AMP-based comprehensive thyroid and lung (CTL) assays, VariantPlex and FusionPlex CTL, with probes covering relevant genes in thyroid and lung cancers. Here, we demonstrate that parallel interrogation of DNA and RNA using VariantPlex and FusionPlex CTL assays, respectively, enables simultaneous detection of known and unknown SNVs, indels, CNVs and fusions. Furthermore, we show that characterization of gene expression, including detection of splice variants, expression imbalances and whole gene expression levels provides orthogonal verification of detected mutations. Our data demonstrate the utility of VariantPlex and FusionPlex CTL assays to simultaneously detect all types of genetic aberrations of clinically relevant genes in thyroid and lung cancer FFPE samples.

Anchored Multiplex PCR enables NGS-based detection of FLT3-ITDs

Marc Bessette, Benjamin Van Deusen, Laura Johnson, Aaron Berlin, Michael Banos, Laura Griffin, Erik Reckase, Joshua Stahl, Abel Licon, Brian A. Kudlow

ArcherDX, Inc., Boulder, CO

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. NGS-based methods often fail to detect FLT3-ITDs, as many variant callers fail to identify these highly variable repeated sequences.

To improve NGS-based detection of FLT3-ITDs, we developed the VariantPlex CoreAML assay, a target enrichment assay based on Anchored Multiplex PCR (AMP™), with probes encompassing the commonly mutated juxtamembrane domain and tyrosine kinase domain 1. We also designed a novel de novo assembly algorithm to assemble resulting sequencing reads within the Archer Analysis bioinformatics pipeline.

To validate the VariantPlex CoreAML assay and Archer Analysis detection algorithm for FLT3-ITD detection, we tested over 20 clinical samples with known FLT3-ITDs and over 2,000 in silico datasets representing the spectrum of known ITDs. Our data showed exceptional sensitivity and specificity in the detection of FLT3-ITDs in both clinical specimens and in silico datasets. Therefore, this AMP-based VariantPlex CoreAML assay in conjunction with the de novo detection algorithm is an accurate method to detect FLT3-ITDs in clinical sample types.

When & Where

Monday, August 8:
  • 3:30pm - 3:55 pm, Afternoon Break and Poster Viewing in the Exhibit Hall (Silverton Ballroom)
Tuesday, August 9:
  • 4:00pm - 4:25 pm, Afternoon Break and Poster Viewing in the Exhibit Hall (Silverton Ballroom)
  • 6:00pm - 7:00 pm, Poster Viewing in the Exhibit Hall (Silverton Ballroom) with Cash Bar.

About ArcherDX

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.

How to contact us


2477 55th Street, Suite 202

Boulder, CO 80301


Phone: (877) 771 1093

Phone: (303) 357 9001

All content © 2018 ArcherDX, Inc.

For Research Use Only. Not for use in diagnostic procedures. For Research Use Only. Not for use in diagnostic procedures.