Archer Reveal ctDNA™ 28

Liquid biopsy mutation profiling with Archer Reveal ctDNA™ 28

Introduction

The Archer® Reveal ctDNA™ 28 Kit for Illumina® is an advanced and user-friendly solution for targeted next generation sequencing (NGS) of circulating cell-free tumor DNA (ccfDNA/cfDNA/ctDNA) from 28 genes commonly found mutated in solid tumor type cancers.

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Anchored Multiplex PCR Enables Sensitive and Specific Detection of Variants in Circulating Tumor DNA by Next-Generation Sequencing, poster presented by ArcherDX at AMP Global, 2017.

The Reveal ctDNA reagent kit preferentially amplifies highly fragmented circulating tumor DNA (ctDNA) over larger genomic fragments (i.e. lysed WBCs) that would otherwise compromise sensitive variant detection. Single-use lyophilized reactions in a convenient 1-day workflow requiring ~1 hour of total hands-on time dramatically simplifies complex NGS library preparation procedures, reduces the chance of operator error and improves overall inter-assay reproducibility.

Powerful and simple-to-use Archer Analysis software ensures sensitive and accurate mutation detection, complete data ownership and in-house data analysis, providing a useful alternative to send-out services that require high sample input volumes and have long turnaround times.

Highlights

  • Relevant fragment captureAnchored Multiplex PCR (AMP™)-based target enrichment preferentially enriches for highly fragmented ctDNA fragments over genomic cell-free DNA (cfDNA) to reduce background and increase detection sensitivity of low-allelic frequency (AF) mutations
  • Single-day library prep — NGS-ready libraries can be prepared in under 8 hours with less than 1 hour of hands-on time, providing sequencing results in as little as 3 days
  • Error correction & quantitative analysis — unique molecular barcode sequences ligated prior to amplification enable duplicate read binning and advanced error correction for confident variant calls
  • Sensitive & robust variant detection — Reliably detect high-AF (5%) variants using as little as 5ng total cfDNA input and low AF (1%) variants from as little as 10ng

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


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and more details!

Product numbers
AB0021 8-reaction Kit
AB0073 48-reaction Kit
AB0022 16-reaction Starter Kit
AB0045 16-reaction Concordance Kit

Protocols

Illumina® Protocol
Illumina® Product Insert

Product Details

Reveal ctDNA 28 targets

ALK
AKT1
AR
BRAF
CTNNB1
DDR2
EGFR
ERBB2 (HER2)
ESR1
FGFR1
HRAS
IDH1
IDH2
KIT
KRAS
MAP2K1
MAP2K2
MET
NRAS
NTRK1
NTRK3
PIK3CA
PDGFRA
RET
ROS1
SMAD4
MTOR
TP53*

*Full exon coverage


Reveal ctDNA 28 Concordance Kit

Now you can do paired multiplex analysis of FFPE and liquid biopsy samples.

The Archer Reveal ctDNA 28 Concordance Kit combines Archer VariantPlex™ with Reveal ctDNA 28 for powerful copy number and variant detection from solid tumors and sensitive mutation detection from ctDNA, respectively. The kit also includes the Archer PreSeq™ DNA QC Assay to prescreen DNA FFPE samples prior to library prep.

Concordance Kit Contents:

  • Reveal ctDNA 28 reagents, 16 reactions
  • VariantPlex reagents, 16 reactions
  • Gene-specific primers, 32 reactions
  • PreSeq DNA QC Assay, 16 reactions
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Performance Specifications

5-300ng

Input cfDNA Required

7 hours

Total Time Required

1 hour

Hands-on Time

198

# of GSP2 Primers

100%

Targeted Bases Covered

>90%

Raw molecular on-target

Illumina®

Sequencing platform

5M

Recommended Read Depth


High Sensitivity

ctDNA comprises only a small fraction of cfDNA. Considering that driver mutations are often low-frequency mutations (AF < 2%), detecting such low-abundance mutations in highly fragmented material can be challenging.

The Reveal ctDNA 28 kit detects relevant ctDNA variants at allelic frequencies (AF) down to 0.1%, demonstrating the high sensitivity of the assay.

Variant 5%* 1%* 0.1%*
NRAS Q61K 5.98 1.62 0.05
NRAS A59T 6.48 0.88 0.17
PIK3CA E545K 7.08 1.03 0.19
EGFR ΔE746—A750 5.88 1.10 0.17
EGFR V769—D770insASV 1.89 0.48 0.05
EGFR L858R 4.69 0.73 0.09
KRAS G12D 7.19 0.69 0.07
Average 5.64 0.96 0.11

The Archer Reveal ctDNA 28 kit was used to create libraries from reference standards comprised of 8 variants at specific allelic frequencies (5%, 1% and 0.1%; HD780, Horizon Discovery) and AF values calculated. *expected AF


Error correction reduces false-positive calls

Low-frequency mutation detection requires increased sequencing depth, which intrinsically results in more sequencing errors and greater noise in the data that leads to false-positive calls. In AMP chemisty, template DNA fragments are ligated to molecular barcodes, which the Archer Analysis bioinformatics platform uses for read provenance to correct for PCR and sequencing errors, thus reducing false-positive calls.

Anchored Multiplex PCR enrichment chemistry corrects for PCR and sequencing errors
Archer Reveal ctDNA 28 kit uses Anchored Multiplex PCR enrichment chemistry to correct for PCR and sequencing errors

Libraries were created with the Archer Reveal ctDNA 28 kit using a 0% AF reference standard, and the number of unique supporting reads with and without error correction were reported. The dotted line indicates the minimum number of unique reads (ten) to call a variant.


Accurate—no matter the input mass

ctDNA abundance in liquid biopsy samples varies across solid tumor types and disease states. The Reveal ctDNA 28 kit enables accurate variant detection across a wide range of input masses, demonstrating assay flexibility that is necessary for liquid biopsy research.

Archer Reveal ctDNA 28 kit delivers accurate variant detection

Libraries were created with the Archer Reveal ctDNA 28 kit using different input masses of the reference standards at 5%, 1% and 0.1% expected AF. Mean AF values across 8 variants in the reference standards were reported above. Dotted lines indicate expected AF.


Simple and fast workflow

AMP is designed with simplicity in mind. The Reveal ctDNA 28 kit uses benchtop-stable lyophilized reagents in single-use 8-tube strips for flexible run sizes and minimun cross-contamination. The workflow comprises serial transfers that total 1 hour of hands-on time for single-day library prep.

Archer Reveal ctDNA 28 kit has a simple and fast workflow

Reproducible variant detection

Consistent assay performance is a hallmark of Archer NGS assays, and the Archer Reveal ctDNA 28 is no exception, even with low AF variants.

Archer Reveal ctDNA 28 kit delivers consistent performance even with low-AF variants

Libraries were created with the Archer Reveal ctDNA 28 kit using reference standards at 5%, 1% and 0.1% expected AF. NRAS A59T AF values for each replicate were reported above. Dotted lines indicate expected AF.


References

  1. E. Crowley, F. Di Nicolantonio, F. Loupakis, A. Bardelli, Liquid biopsy: monitoring cancer-genetics in the blood. Nat Rev Clin Oncol. 10, 472–484 (2013).
  2. L. A. Diaz, A. Bardelli, Liquid biopsies: genotyping circulating tumor DNA. J. Clin. Oncol. 32, 579–586 (2014).
  3. A. Esposito, C. Criscitiello, M. Locatelli, M. Milano, G. Curigliano, Liquid biopsies for solid tumors: Understanding tumor heterogeneity and real time monitoring of early resistance to targeted therapies. Pharmacol. Ther. 157, 120–124 (2016).
  4. M. Gerlinger et al., Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med. 366, 883–892 (2012).
  5. Z. Piotrowska et al., Heterogeneity Underlies the Emergence of EGFRT790 Wild-Type Clones Following Treatment of T790M-Positive Cancers with a Third-Generation EGFR Inhibitor. Cancer Discovery. 5, 713–722 (2015).
  6. M. Holdhoff, K. Schmidt, R. Donehower, L. A. Diaz, Analysis of circulating tumor DNA to confirm somatic KRAS mutations. J. Natl. Cancer Inst. 101, 1284–1285 (2009).
  7. M. Murtaza et al., Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature. 497, 108–112 (2013).
  8. L. De Mattos-Arruda et al., Circulating tumour cells and cell-free DNA as tools for managing breast cancer. Nat Rev Clin Oncol. 10, 377–389 (2013).
  9. L. A. Diaz et al., The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers. Nature. 486, 537–540 (2012).

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How to contact us

Address

2477 55th Street, Suite 202

Boulder, CO 80301

Phone

Phone: (877) 771 1093

Phone: (303) 357 9001

All content © 2017 ArcherDX, Inc.

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