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NGS Assays Purpose-Built for Revolutionary Mutation Calling

ArcherDX, Inc. at AGBT 2015

Advances in Genome Biology and Technology meeting, 2015

February 25th-28th

Marco Island, Florida

To address the existing bottlenecks of using NGS in translational research, we’ve created a line of innovative products that are 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.

Archer™ NGS assays are purpose-built for clinical research

Robust platform to enrich targeted RNA or DNA

Detect known and unknown regions by NGS

Scalable cost- and labor-effective workflow

Comprehensive time-saving automated sequence analysis

Presentations

Thon de Boer, Product Manager - Software, ArcherDX, Inc.

Beyond fusion-calling: SNPS, InDels and CNVs - Archer Analysis software demonstration

Thon de Boer, Product Manager - Software, ArcherDX, Inc.

Thursday, February 26

5:15-7:15 pm

Marco Island Hilton

Dinner provided

Brian Kudlow, Senior R&D Manager/Lead Scientist, ArcherDX, Inc.

Sequencing That Counts – Applications of Anchored Multiplex PCR in the Detection of Copy Number Variation

Brian Kudlow, Senior R&D Manager/Lead Scientist, ArcherDX, Inc.

Friday, February 27

4:00 pm

Islands Ballroom


Posters

Brian Kudlow, Senior R&D Manager/Lead Scientist, ArcherDX, Inc.

See Brian's Poster

Development and Validation of a Directed, Rapid and Highly-Multiplexed Assay for the Detection of Copy Number Variations in Clinical Samples

Brian Kudlow, Senior R&D Manager/Lead Scientist, ArcherDX, Inc.

Abstract

Copy number variation (CNV), either amplification of oncogenes or deletions of tumor suppressor genes, is a common mode of gene de-regulation in cancer. Several techniques, including comparative genomic hybridization (CGH) and quantitative PCR (qPCR) have been employed to determine copy number; however, none of these methods are amenable to high-throughput, directed detection of CNVs...

We have developed a directed next generation sequencing (NGS)-based method to rapidly and quantitatively measure copy number of tens and potentially hundreds of genes simultaneously. This complete workflow, using the Archer by Enzymatics LyoFire DNA Fragmentation and Anchored Multiplex PCR (AMP) reagents, allows dozens of samples to be processed in about 6 hours. By ligating a molecular barcode to randomly fragmented input DNA, and then simultaneously enriching for several regions of each target gene through AMP, we are able to accurately measure the relative copy number of each target gene in test samples by counting unique molecular barcodes associated with each target region.

We have validated our methodology with a 25-gene panel on a subset of NCI-60 cell lines by comparing our copy number measurements to those determined by both CGH and qPCR. Both orthogonal methods showed extremely strong correlation with our NGS-based method. Multiplexing as many as 150 samples on a single MiSeq run permitted detection of CNVs, both amplifications and deletions, of 2X magnitudes (and often lower) at extremely high confidence, indicating that this panel is amenable to highly multiplexed screens of potentially hundreds of samples. Furthermore, we demonstrate that our NGS-based CNV detection workflow and analysis is compatible with DNA extracted from formalin-fixed, paraffin embedded (FFPE) samples, suggesting that this system could be adapted for use in clinical applications.

Abel Licon, Group Lead NGS Software Apps ArcherDX, Inc.

See Abel's Poster

Archer FusionPlex NGS Data Analysis Algorithms and Methods: Hypothesis-free gene fusion detection

Abel Licon, Group Lead NGS Software Apps ArcherDX, Inc.

Abstract

The Archer™ Analysis pipeline contains hypothesis-free fusion detection algorithms and methods that are built to leverage the advantages of the Archer™ Anchored Multiplex PCR (AMP) based FusionPlex assay. Compatible with Ion Torrent and Illumina, the pipeline can accurately detect known fusions, identify novel fusions, and identify false fusions that result from AMP chemistry. The pipeline addresses many known issues that generally result from high throughput targeted amplicon sequencing.

The AMP FusionPlex assay utilizes randomly generated 8-mers as molecular barcodes, or MBCs. Because each unique starting molecule of RNA is tagged with an MBC, the analysis method can measure PCR duplication rates by counting unique MBCs as opposed to using unique alignment positions. Furthermore, the pipeline leverages PCR replicates in order to detect and remove non-systematic sequencing error, which is crucial in determining the final chimeric protein. The pipeline also maximizes performance and specificity by using a waterfall approach to explain the simplest non-chimeric reads before attempting to map chimeric reads to the entire genome, an optimization that cannot be done using non-targeted approaches. The pipeline leverages the Quiver™ database, which is an aggregation of several popular gene fusion resources. Quiver™ is used to mark fusions as known or novel, giving confidence to known fusions and providing guidance for validating novel ones. The consensus sequence provided by the pipeline gives single base pair resolution which is used to predict the translational frame of the final protein product, a level of resolution not possible with non-sequencing approaches. For validation, known fusion positive and fusion negative FFPE samples and cell-lines have been analyzed with the Archer Analysis pipeline. In order to increase accessibility, the pipeline provides a parallel, scalable and user friendly web-interface to the gene fusion detection pipeline, removing the need for in-house informatics experts to analyze raw high-throughput sequencing data.

Abel Licon, Group Lead NGS Software Apps ArcherDX, Inc.

See Brady's Poster

Optimization of nested PCR conditions to improve target coverage uniformity in Archer VariantPlex BRCA panel

Brady Culver, Senior R&D Manager/Lead Scientist, ArcherDX, Inc.

Abstract

ArcherDX, Inc’s proprietary Anchored Multiplex PCR™ (AMP) chemistry for NGS applications is a demonstrated robust tool for detecting both known and unknown fusions. However, characteristics associated with AMP transcend RNA assays and make it a valuable tool for DNA sequencing as well. These characteristics include: the ability to identify unique starting molecules and consolidate sequencing information from PCR duplicates, low input DNA requirements, and applicability to both Illumina and Ion Torrent sequencing platforms.

Additionally, the enzymatic DNA fragmentation method used in Archer VariantPlex assays generates a broad sequenceable fragment size distribution, ranging from less than 100bp to more than 1 kilobase in length. The presence of longer fragments facilitates read mapping over repetitive or conserved regions in the genome. The first Archer DNA assay for wide spread release is the Archer VariantPlex™ BRCA1/2 Panel. Inherited harmful BRCA1/2 mutations substantially increase one’s lifetime risk of developing cancer, especially breast and ovarian cancer. The VariantPlex BRCA1/2 Panel design covers all coding and non coding exons in RefSeq annotated transcript models for these genes. In an effort to further improve panel performance, we investigated alterations to PCR cycling conditions for enhancement of target coverage uniformity. Our data shows that a balance must be struck between primer performance uniformity and the portion of total reads that are on-target. Despite losses in on-target reads, fewer reads were required to cover all targeted bases than conditions with higher on-target percent. In addition, coverage and sensitivity were universally improved by utilizing a new polymerase. These changes are being made across all Archer NGS kits. Finally, we demonstrate the performance of this panel and our variant calling software by correctly identifying the known BRCA1 or BRCA2 mutations present in 10 different samples from Coriell.

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