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Precise Genomic Definition

A major challenge facing many laboratories lies in sourcing reference standards that reflect patient samples.  Historically, a wide range of types of material has been used, including synthetic DNA such as oligonucleotides or plasmids, cell culture lines, and patient derived materials, none of which are ideal, as outlined in the table below.

 Defined copy numberDefined Allele BurdenStoichiometric Multiplexing CapabilityRepresents Tumor Samples
Genome ComplexityCompromised DNA (FFPE)Tumor Cell Genetics
Synthetic oligos

Cell line mixtures

Primary tissue samples

Synthetic oligos spiked into cell DNA

HDx™ Reference Standards


Gene Mutation
B-Raf V600E 25% 5% 3.5% 1%
B-Raf V600K 50% 5% 1% --

Patient derived material is considered by many to be the gold standard, however the high degree of variability between and even within samples is a problem when used as a reference material.  This issue is compounded by the limited supply of any given sample, particularly in the case of rare tumors.

Synthetic reference standards such as oligonucleotides and plasmids tend to amplify more easily than equivalent template from cellular sources, which can create false confidence in molecular assay workflows. Spiking such material into cellular DNA doesn’t solve the problem, as the issue of achieving a biologically relevant copy number then emerges.

Horizon Diagnostics works with highly characterized parental cell lines which have been single cell cloned and banked accordingly, and form the basis for the various engineered mutant cell lines.  By having such closely matched parental origins we are able to demonstrate both the genetic complexity and the relative isogenicity found in tumors.

Horizon Diagnostics uses its proprietary GENESIS™ genome engineering platform to precisely engineer mutations into highly characterized parental cell lines, thereby allowing these “normal” cell lines to have fully defined cancer mutations at the endogenous locus.  After generation, Horizon Diagnostics validates cell lines in order to understand the sequence and copy number of each locus.  This results in perfectly engineered heterozygotes that endogenously contain the mutation of interest.

In order to further mimic real patient samples Horizon Diagnostics has developed expertise around blending materials, either as purified gDNA or as cells for embedding in FFPE.  This has allowed us to generate, at very precise allelic frequencies, mutant/ wild type mixtures ranging between 1% and 50% mutant, verified using digital PCR.

In addition to the precise specifications associated with each product, HDx™ Reference Standards are renewable and highly reproducible, making then perfectly suited to assessing, managing and mitigating variability in molecular assays.

HDx™ Quantitative Molecular Reference Standards

Genomic DNA Dilutions

Tumour Sample Heterogeneity - HDx™ Reference Standards mimic the heterogeneity seen in real patient samples


Phenotype Wildtype



Genotype Wildtype 0.5% - 1%
25% - 50%
50% - 100%

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