Quantitative Multiplex Applications

Horizon has developed a Quantitative Multiplex HDx Reference Standard containing 8 engineered mutations and 3 three endogenous mutations, each calibrated by digital PCR to a chosen allelic frequency ranging between 0.8% and 24%.  This standard has been designed to highlight inconsistencies of results, allowing scientists to better understand their workflows and improve robustness over time.

Key Features:

  • Complete AmpliSeq™, TruSeq™ and TruSight™ gene coverage
  • NGS dataset available for download
  • Quantitative reference standard, enabling objective assessment of multiplex assay performance
  • 11 different validated cancer genotypes, supported by additional validated mutations present in oncology relevant alleles
  • Copy number and allelic frequency are precisely controlled, and validated using digital PCR.
  • Available as genomic DNA and FFPE

Case Study:

Data has shown that while NGS platforms generally perform well, it is common to find inconsistent allele detection between different users and different workflows, even when they are using the same platform.  The example below, taken from multiple laboratories and multiple platforms highlights this variability.

Performance of Ion Torrent™

Source:Horizon DiscoveryPartner APartner BPartner CPartner D

Platform:

QX100™ Droplet Digital PCR

Ampliseq Cancer Panel

Ampliseq Cancer Hotspot Panel v2

Ampliseq Cancer Panel v2 (Average of 8 runs)

Sequencing Depth

N/A

3000-4000x

Average 5000x

2000x

GeneMutationObserved mutant ratio

BRAF

V600E

10.2

9.9

9.1

10.3

KIT

D816V

10.4

10.0

11.0

10.1

EGFR

ΔE746 - A750

2.0

2.3

Not detected

Not detected

EGFR

L858R

2.7

2.7

2.1

2.4

EGFR

T790M

0.9

0.8

Not detected

Not detected

EGFR

G719S

24.4

23.7

23.1

24.8

KRAS

G13D

16.1

16.3

12.35

15.5

KRAS

G12D

5.0

5.2

Not detected

5.1

NRAS

Q61K

12.8

9.0

12.7

12.6

PIK3CA

H1047R

18.6

16.7

16.8

17.9

PIK3CA

E545K

8.9

3.2

8.4

8.8

Lower allelic frequencies were not always picked up and increased sequencing depth did not guarantee better outcomes.  The Quantitative Multiplex HDx Reference Standard helps to highlight these unintended outcomes, allowing scientists to better understand and control their workflow over time

view our Quantitative Multiplex Products

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