Multiplex I cfDNA in Synthetic Matrix II

The most flexible reference standard for cfDNA recovery

This product is an optimized version of HD816 - Multiplex I cfDNA Reference Standard Set in Synthetic Plasma and HD817 - Multiplex I cfDNA Reference Standard Set in Synthetic Matrix designed to be compatible with the most commonly used extraction kits - including bead and column based methods (see supporting data) - making it the most flexible reference standard for cfDNA recovery.

Multiplex I cfDNA Reference Standard Set in Synthetic Matrix II contains 8 onco-relevant mutations to measure your extraction efficiency and assay performance. As an optimal synthetic matrix that enables extraction recovery of cfDNA by multiple purification methods, its clinically relevant mutations in an artificial matrix give you full control of your cfDNA workflow, reducing the likelihood of a false negative or positive due to incorrect assay loading.

Extraction Platform Capability

Cat no.	Description	Extraction platform
		Column based		Column/Bead	Bead based
		QiAamp Circulating Nucleic Acid Kit	Macherey Nagel Nucleosnap Plasma Kit	Qiagen Min Elute cfDNA Kit	MagMax cfDNA Isolation Kit	Maxwell RSC cfDNA Isolation Kit
HD816	Multiplex I cfDNA in synthetic plasma	✓	Not recommended	✓	Not recommended	✓
HD817	Multiplex I cfDNA in synthetic matrix	✓	Not recommended	✓	Not recommended	✓
HD917	Multiplex I cfDNA in synthetic matrix II	✓	✓	✓	✓	✓

The Multiplex I cfDNA Reference Standard Set in artificial matrix covers multiple engineered single nucleotide variants (SNVs/SNPs) with 8 mutations at 5%, 1%, and 0.1% allelic frequencies.

For information on specific gene copies detected in each product batch, download the product insert from the resources tab.

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: Synthetic matrix II

Expected DNA yield: ≥120 ng DNA per mL of product using the QIAamp® Circulating Nucleic Acid Kit with 2 mL extraction input

Product information

Fragment size: 170bp

Unit size: 400ng per 1ml (4 vials total)

Extraction platform compatibility

Product	Extraction platform
	Column based		Column/Bead	Bead Based
	QIAamp Circulating Nucleic Acid Kit	Macherey Nagel NucleoSnap Plasma kit	QIAgen MinElute cfDNA kit	MagMAX cfDNA isolation kit	Maxwell RSC cfDNA isolation kit
Synthetic Matrix II

Verified mutations

HD917 component expected allelic frequencies (%)

EGFR

Gene	Variant (AA)	CDS mutation	GRCh38 co-ordinates*	Cosmic ID	HD912 5%	HD913 1%	HD914 0.1%	HD915 100% WT
EGFR	T790M	c.2369C>T	7:55181378	COSV51765492	5	1	0.1	0
EGFR	delE746-A750	c.2235_2249del	7:55174772-55174786	COSV51765119	5	1	0.1	0
L858R	c.2573T>G	7:55191822	COSV51765161	5	1	0.1	0
EGFR	V769-D770insASV/A767_V769dup	c.2300_2308dup	7:55181317-55181318	COSV51766549	5	1	0.1	0
NRAS	Q61K	c.181C>A	1:114713909	COSV54736310	6.3	1.3	0.13	0
NRAS	A59T	c.175G>A	1:114713915	COSV54743124	6.3	1.3	0.13	0
PIK3CA	E545K	c.1633G>A	3:179218303	COSV55873239	6.3	1.3	0.13	0
KRAS	G12D	c.35G>A	12:25245350	COSV55497369	6.3	1.3	0.13	0

*GRCh37 variant co-ordinates are shown in the Exome sequencing data

Presence confirmed in parental cell line: EGFR G719S (20% allelic frequency)

Exome sequencing data: The cfDNA in buffer format of this product (HD780) has been verified by whole exome sequencing. To download the full data set, in variant caller format (VCF), please visit our exome sequencing page. This data set provides information on variants in the parental cell lines which may be detected when performing NGS analysis of this product.

General information

Shipping conditions: Ambient (shipped with gel packs to ensure item maintains room temperature storage during transport)

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)

We have summarized the extraction efficiency of Matrix I (HD816/HD817) and Matrix II (HD917) using a range of the most used extraction kits.

Introduction

The analysis of cell-free circulating nucleic acids is becoming a powerful tool for the provision of personalized medicine to cancer patients. Clinical samples have extremely low quantities of cfDNA so it is important to ensure that cell-free DNA extraction from plasma is as efficient as possible. Cell-free DNA reference standards allow us to monitor the performance of extraction techniques, as well as downstream molecular assays. The use of a synthetic plasma to make reference standards is often preferred over human plasma. This is due to its stability, lot-to-lot consistency, and absence of interfering substances. Not all synthetic plasmas are compatible with the existing range of extraction methods and molecular assays. Here we demonstrate the compatibility of a cell-free DNA reference standard in synthetic plasma with a range of different extraction techniques, including the two most popular approaches: column-based extraction and magnetic bead-based extraction.

Method

Our Matrix I (HD817) and Matrix II (HD917) products consist of cfDNA containing known variants at specific allelic frequencies spiked into synthetic plasmas. The spiked-in cfDNA was extracted from Matrix I and Matrix II using five popular cfDNA extraction kits, which include the two most used extraction technologies: bead based and column-based chemistry.

Extraction Kits used in this study:
ThermoFisher MagMAX™ Cell-Free DNA Isolation Kit (#A29319)
Qiagen MinElute ccfDNA Kit (#52204)
Qiagen QIAamp Circulating Nucleic Acid Kit (#55114)
Macherey Nagel NucleoSnap DNA Plasma (#740300)
Maxwell RSC ccfDNA Plasma Kit (Cat #AS1480)

Extraction efficiencies with 5 cfDNA extraction kits

Result

Matrix I (HD816/HD817) demonstrated satisfactory efficiencies in three of the kits tested, however a low yield is recovered when using Machery Nagel Nucleosnap kit, and no cfDNA is recovered using the ThermoFisher MagMAX extraction kit. On the other hand, Matrix II (HD917) demonstrated higher efficiencies and compatibility with the most common extraction kits used for cfDNA isolation, including MagMAX and Machery Nagel Nucleosnap.

Comparison of extraction efficiencies with five cfDNA extraction kits when spiking cfDNA into Matrix I (HD817) or Matrix II (HD917)

Matrix II HD917 - extraction efficiencies with five cfDNA extraction kits

Results demonstrate that Matrix II (HD917) has a higher compatibility with the most common extraction kits available for cfDNA, including bead and column based methods.