- Molecular reference standards
- Multiplex I cfDNA Reference Standard Set
Multiplex I cfDNA Reference Standard Set
The Multiplex I cfDNA Reference Standards are highly-characterized, biologically-relevant reference materials used to assess the performance of cfDNA assays that detect somatic mutations, including quantitative PCR, next-generation sequencing, and other novel assays
High throughput platforms such as Next generation sequencing (NGS) and quantitative PCR (qPCR) platforms give researchers much greater power when profiling cfDNA samples. However, the use of these presents challenges surrounding sample variability, platform bias, and potential failure to detect biomarkers. Horizon’s cfDNA products are all derived from human cell lines, and are fragmented to an average size of 160 bp to closely resemble cfDNA extracted from human plasma.
This format ensures that they may applied to a wide-range of cfDNA NGS and qPCR assays including high-throughput platforms such as NGS and qPCR give researchers greater power when profiling cfDNA samples. Horizon has developed the cfDNA Reference Standard range to support the development and continued validation of cfDNA assays.
The Multiplex I cfDNA Reference Standard Set covers multiple engineered single nucleotide variants (SNVs/SNPs) with 8 mutations at 5%, 1%, and 0.1% allelic frequencies. Batch-specific copy number values measured via ddPCR are provided with each Set.
With this product you are able to:
- Analyze the sensitivity and specificity of your assay
- Gain certainty of the limit of detection and limit of quantification
- Optimize and validate new cancer panels and routinely monitor the performance of your assay
Technical data
Format: cfDNA
Genes covered: EGFR, KRAS, NRAS, PIK3CA
Cosmics: 1,500+
Allelic frequencies: 5%, 1%, 0.1% and 0% (100% wild type)
Buffer: Tris-EDTA (10mM Tris-HCl, 1mM EDTA), pH 8.0
Product information
Fragment size: 160 bp
Unit size: 350 ng per vial
Concentration: 20 ng/µl
Verified mutations: The Multiplex cfDNA Reference Standard has been manufactured using multiple engineered cell lines and mixed to generate multiple Allelic Frequencies in multiplex. Two of the four products in this set (HD776 and HD777) have been verified by whole exome sequencing. The NGS determined allelic frequency and read depths for HD777 are reported in the table below.
Multiplex cfDNA 0% (HD776) | ||||||||
---|---|---|---|---|---|---|---|---|
Gene | Variant (AA) | CDS mutation | GRCh38 co-ordinates | Legacy ID | Genomic mutation ID | Expected allelic frequency (%) | NGS allelic frequency (%) | NGS read depth |
EGFR | L858R | c.2573T>G | 7:55191822 | COSM6224 | COSV51765161 | 0.00% | b | b |
EGFR | ΔE746-A750 | c.2235_2249del | 7:55174771 | COSM6223 | COSV51765119 | 0.00% | b | b |
EGFR | T790M | c.2369C>T | 7:55181378 | COSM6240 | COSV51765492 | 0.00% | b | b |
EGFR | A767_V769dup | c.2300_2308dup | 7:55181317 | COSM12376 | COSV51766549 | 0.00% | b | b |
KRAS | G12D | c.35G>A | 12:25245350 | COSM521 | COSV55497369 | 0.00% | b | b |
NRAS | Q61K | c.181C>A | 1:114713909 | COSM580 | COSV54736310 | 0.00% | b | b |
NRAS | A59T | c.175G>A | 1:114713915 | COSM578 | COSV54743124 | 0.00% | b | b |
PIK3CA | E545K | c.1633G>A | 3:179218303 | COSM763 | COSV55873239 | 0.00% | b | b |
Multiplex cfDNA 5% (HD777) | ||||||||
---|---|---|---|---|---|---|---|---|
Gene | Variant (AA) | CDS mutation | GRCh38 co-ordinates | Legacy ID | Genomic mutation ID | Expected allelic frequency (%) | NGS allelic frequency (%) | NGS read depth |
EGFR | L858R | c.2573T>G | 7:55191822 | COSM6224 | COSV51765161 | 5.00% | 4.50% | 422 |
EGFR | ΔE746-A750 | c.2235_2249del | 7:55174771 | COSM6223 | COSV51765119 | 5.00% | 3.47% | 432 |
EGFR | T790M | c.2369C>T | 7:55181378 | COSM6240 | COSV51765492 | 5.00% | 4.38% | 1690 |
EGFR | A767_V769dup | c.2300_2308dup | 7:55181317 | COSM12376 | COSV51766549 | 5.00% | 3.78% | 2064 |
KRAS | G12D | c.35G>A | 12:25245350 | COSM521 | COSV55497369 | 5.00% | 5.53% | 380 |
NRAS | Q61K | c.181C>A | 1:114713909 | COSM580 | COSV54736310 | 5.00% | 6.91% | 622 |
NRAS | A59T | c.175G>A | 1:114713915 | COSM578 | COSV54743124 | 5.00% | 5.66% | 618 |
PIK3CA | E545K | c.1633G>A | 3:179218303 | COSM763 | COSV55873239 | 5.00% | 4.81% | 499 |
Multiplex cfDNA 1% (HD778) | ||||||||
---|---|---|---|---|---|---|---|---|
Gene | Variant (AA) | CDS mutation | GRCh38 co-ordinates | Legacy ID | Genomic mutation ID | Expected allelic frequency (%) | NGS allelic frequency (%) | NGS read depth |
EGFR | L858R | c.2573T>G | 7:55191822 | COSM6224 | COSV51765161 | 1.00% | Not tested | Not tested |
EGFR | ΔE746-A750 | c.2235_2249del | 7:55174771 | COSM6223 | COSV51765119 | 1.00% | Not tested | Not tested |
EGFR | T790M | c.2369C>T | 7:55181378 | COSM6240 | COSV51765492 | 1.00% | Not tested | Not tested |
EGFR | A767_V769dup | c.2300_2308dup | 7:55181317 | COSM12376 | COSV51766549 | 1.00% | Not tested | Not tested |
KRAS | G12D | c.35G>A | 12:25245350 | COSM521 | COSV55497369 | 1.00% | Not tested | Not tested |
NRAS | Q61K | c.181C>A | 1:114713909 | COSM580 | COSV54736310 | 1.00% | Not tested | Not tested |
NRAS | A59T | c.175G>A | 1:114713915 | COSM578 | COSV54743124 | 1.00% | Not tested | Not tested |
PIK3CA | E545K | c.1633G>A | 3:179218303 | COSM763 | COSV55873239 | 1.00% | Not tested | Not tested |
Multiplex cfDNA 0.1% (HD779) | ||||||||
---|---|---|---|---|---|---|---|---|
Gene | Variant (AA) | CDS mutation | GRCh38 co-ordinates | Legacy ID | Genomic mutation ID | Expected allelic frequency (%) | NGS allelic frequency (%) | NGS read depth |
EGFR | L858R | c.2573T>G | 7:55191822 | COSM6224 | COSV51765161 | 0.10% | Not tested | Not tested |
EGFR | ΔE746-A750 | c.2235_2249del | 7:55174771 | COSM6223 | COSV51765119 | 0.10% | Not tested | Not tested |
EGFR | T790M | c.2369C>T | 7:55181378 | COSM6240 | COSV51765492 | 0.10% | Not tested | Not tested |
EGFR | A767_V769dup | c.2300_2308dup | 7:55181317 | COSM12376 | COSV51766549 | 0.10% | Not tested | Not tested |
KRAS | G12D | c.35G>A | 12:25245350 | COSM521 | COSV55497369 | 0.10% | Not tested | Not tested |
NRAS | Q61K | c.181C>A | 1:114713909 | COSM580 | COSV54736310 | 0.10% | Not tested | Not tested |
NRAS | A59T | c.175G>A | 1:114713915 | COSM578 | COSV54743124 | 0.10% | Not tested | Not tested |
PIK3CA | E545K | c.1633G>A | 3:179218303 | COSM763 | COSV55873239 | 0.10% | Not tested | Not tested |
a – Not detected - did not meet detection threshold, below LOD
b – 0.00% expected VAF, not detected by variant caller
General information
Storage: 4°C
Expiration: See all product shelf life information
Quality control
Fragmentation size: D1000 DNA ScreenTape assay
Allelic frequency: Droplet Digital™ PCR
Quantification: Qubit dsDNA BR Assay (post-fragmentation)
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