HORIZON DIAGNOSTICS

Base-SeqHDx™ Reference StandardsSanger and qPCR Sequencing

Reference StandardsIdentify and Control Variability Every Day

Routinely monitor the performance of your workflows and assays with independent external controls

• Establish your absolute limit of detection for every mutation

• Determine the specificity and sensitivity of your assay with ease

• Ensure confidence in your assay results

tfew Horizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom

+ 44 (0)1223 655 580 (UK) or +1 800 860 1567 (USA)+ 44 (0)1223 655 581info@horizondx.comwww.horizondx.com

For Research Use Only

HDx Reference Standards Identify and Control Variability Every Day

Workflow VariabilityThe ProblemDNA sequencing data generated using the Sanger method is often limited by poor quality in the first 15-40 bases of the sequence (due to primer binding) and by a maximum possible read length of about 700-900 bases, with a significant deterioration in quality thereafter.

Further to this, use of Sanger sequencing to detect nucleotide polymorphisms requires that secondary alleles are present in at least 20% frequency, leaving a potential for false negative results in samples around or below this frequency.

The SolutionTo identify and control this variability, Horizon Diagnostics has developed multiple formats of Reference Standards to ensure the accuracy of your workflow every day:

• Genomic DNA for your assay

• Formalin-Fixed, Paraffin-Embedded (FFPE) Sections for your workflow

FFPE Sections (Pre-Analytical)

Routinely monitor your workflow performance

• Test the full integrity of your molecular assay workflow (extraction, quantification, molecular testing and data analysis)

• Formalin-Fixed, Paraffin-Embedded

Genomic DNA (Analytical)

Routinely monitor your assay performance

• Identify the analytical specificity and sensitivity of your assay

• Over 100 mutations available

Matched Wild-Type DNA (Analytical)

True negative control

• Use for allelic frequency dilutions

• Determine the absolute Limit of Detection

Pre-Analytical

DNA Extraction

DNA Quantitation

Sequencing/ qPCR (Analytical)

There is the potential therefore for these challenges to cause assay errors. This is highlighted in a recent worldwide EGFR proficiency testing scheme, the External Quality Assessment (EQA) 2014 [1], which over 20% of participating laboratories failed.

Laboratories often assume that a failed assay is as a result of a failed analytical step; however there are multiple sources of variability in any molecular assay workflow (see diagram) and frequently root cause is a problem with DNA extraction or issues with DNA quantification leading to the assay being carried out with an insufficient amount of DNA.

Product FormatQuantity 1 FFPE Section

Extractable DNA 400ng - 700ng

1 Extractable use per vial

Product FormatDNA Quantity 1μg

Allelic frequency 50%

100 Daily uses per vial

• Generate the absolute Limit of Detection (LOD) with Mutant and Wild Type DNA

• Allelic frequency can be modified as required.

• Independent assay validation & routine monitoring of analytical performance for both specificity & sensitivity

• Over 100 mutations available

Genomic DNA Routinely monitor your Assay

Product FormatDNA Quantity 5μg

Concentration 50ng/μl

500 Daily uses per vial

• Matched Wild Type DNA for over 40 genotypes

• Determine the absolute limit of detection (LOD)

Matched Wild Type DNATrue negative Standard

• Range of precisely validated allelic frequencies [1%, 5%, 20% and 50%]

• Standards mimic cancer patient genetics & processed tissue sample quality

• Test the full integrity of a molecular assay workflow [extraction, quantification, molecular testing & data analysis]

Formalin-Fixed, Paraffin-Embedded (FFPE)Routinely monitor your Workflow

Available Formats

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EGFR Sample Tested

EGFR Genotyping Errors

Figure 1: EQA results, 2014

EGFR Genotyping Errors

Ensure the accuracy of your workflow with HDx Reference StandardsVisit www.horizondx.com/Base-Seq

Ensure the consistency, specificity and sensitivity of your Sanger and qPCR assays with independent external controls Key features

• Precisely validated for copy number and allelic frequency using digital PCR

• Standards mimic cancer patient genetics

• FFPE format allows for authentic, in process control from extraction to reporting, whilst DNA format enables robust assessment of limit of detection

• Highly consistent and renewable resource

Applications

• Full in-process control

• Method testing e.g. DNA extraction, DNA quantification

• Assay/ platform optimization

• Staff training

Mutations available

tfew Horizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom

+ 44 (0)1223 655580+ 44 (0)1223 655581info@horizondiscovery.comwww.horizondiscovery.com

Validation Test AssayAllelic Frequency Droplet Digital PCR™Gene Copy Number Droplet Digital PCR™Genotype Sanger Sequencing of locus specific PCRQuantification Spectrophotometric assay (DNA)/Quantifluor (FFPE-extracted DNA)

These standards are manufactured under ISO:13485 and ISO:9001 and independently validated with a broad range of molecular assays to ensure consistency

References1 Patton et al., Assessing standardization of molecular testing for non-small-cell lung cancer: results of a worldwide external quality assessment (EQA) scheme for EGFR mutation testing. British Journal of Cancer, 2014, 111(2):413-20.

BRAF (15)

EGFR (24)

IDH1 & IDH2 (6)

KRAS (29)

NRAS (15)

PI3KCA (10)

Other (27)

BRAF (15)

EGFR (24)

IDH1 & IDH2 (6)

KRAS (29)

NRAS (15)

P13KCA (10)

Other (27)

Base-Seq HDx™ Reference Standards, Sanger and qPCR Sequencing 2015 v-01