cfDNA - Frequently Asked Questions

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What are cfDNA HDx Reference Standards?

A major clinical goal, especially for oncology, is to achieve non-invasive disease assessment and progression monitoring. The analysis of cell free DNA (cfDNA), also known as circulating tumor DNA (ctDNA) is currently the most promising technical advances to provide an alternative to tumor biopsy. Translating the application of ctDNA detection into the clinic requires stringent procedures to enable both accurate and precise measurements. Meanwhile ctDNA detection methodologies face new challenges due to the low quantity of highly-fragmented material available from biofluids. Analysis of ctDNA is often intended to identify very low allele frequency mutations, including 0.1% and lower, that push the detection limits of current technologies. Until now, the availability of reference standards or a universal control to assess cfDNA/ctDNA analytical workflows has broadly been limited to variable clinical specimens.

In response to this need, Horizon has developed the cfDNA Reference Standard range to support the innovation and standardization of this valuable technology. Derived from engineered human cell lines, the reference standards are provided as fragmented human genomic DNA (average size 160 bp) resembling cell tumor DNA derived from human plasma (see Figure 1). The reference standards are provided at allelic frequencies down to 0.1% across a range of variants with a matched wild type. Copy number concentrations and expected allelic frequencies of each product are provided, ideal to support the breath and quality of data you need for validation. These reference standards will help to drive forward the development, validation and demonstration of the performance of cfDNA assays and platforms on a routine basis.

Figure 1: Example trace of the fragment sizes collected by D1000 DNA ScreenTape assay, comparing cfDNA HDx Reference Standards (Red and Green traces) to cfDNA extracted from human plasma (Blue trace). cfDNA from human plasma was provided by CareDx, Inc. Leftmost peaks - internal marker for the assay. Rightmost peaks - fragmented materials. 

 

Why do I need cell free DNA HDx Reference Standards for my workflow?

Using cell free DNA HDx Reference Standards will enable you to:

  • Monitor the performance of your cfDNA assay
  • Optimize and validate your cfDNA assay
  • Challenge the limit of detection of analytical platform used for cfDNA analysis
  • Identify cfDNA platform variability
  • Routinely monitor the performance of cfDNA assay and platform
  • Streamline operator training

 

How are cfDNA HDx Reference Standards different from other circulating tumor DNA controls?

In molecular biology, plasmid DNA has proved to be a vital tool for many applications. However, when developing or validating circulating tumor DNA assays (also known as cfDNA assays) which will ultimately measure fragmented human genomic DNA samples, plasmid and synthetic DNA are not  ideal control samples. Laboratories should be cautious in adopting plasmids as controls for respective assays as they do not represent the true genomic complexity of human tumor samples. Therefore the results achieved with controls derived from plasmids may not be robust.

Read more about the benefits of HDx Reference Standards over plasmids here.

 

How are cfDNA HDx Reference Standards fragmented?

Genomic DNA with mutation extracted from engineered cell line is accurately and precisely sheared using mechanical shearing method. Horizon has also developed optimized Standard Operating Procedures (SOPs) to ensure consistency.

 

Do you perform end repair after shearing?

Horizon does not perform end repair steps. However, users performing end repair and A-addition steps as part of library preparation are encouraged to perform similar steps on the cfDNA reference standards.

 

Have you tried any other methods of creating cfDNA standards?

Horizon has previous experience performing enzymatic digestion to fragment genomic DNA. However this method results in a less reproducible production process and inconsistent fragmentation due to lot-to-lot variability. Therefore Horizon has opted to use its proprietary mechanical shearing methods to reproducibly manufacture cfDNA Reference Standards.

 

What is the range of fragment distribution?

cfDNA HDx Reference Standards are analysed using Next-gen sequencing 12 capillary array Fragment Analyser (Advanced Analytical Systems) and D1000 ScreenTape assay on TapeStation (Agilent Tech). The figure and table below shows cfDNA HDx Reference Standards are sheared into short fragments of up to 400bp and around 60% of the product contains fragments of 100bp to 250bp resembling circulating cell free DNA. The figure also demonstrate consistent fragmentation sizes between each part of the set.

The Fragment analysis was performed by third party on a single batch of Multiplex I cfDNA Reference Standard Set. TapeStation analysis was performed in-house as part of the QC measures.

 Fragment analyser
TapeStation
 

 

10bp to 500bp

100bp to 250bp

35bp to 500bp

100bp to 250bp

 

% of total

Avg size (bp)

% of total

Avg size (bp)

% of total

Avg size (bp)

% of total

Avg size (bp)

Average

100

149

60

161

97

159

62

164

CV (%)

0.1

0.8

2.4

0.8

0.8

2.9

2.1

4

Note: A region between 10-500bp on Fragment analyser and 35-500bp on TapeStation were set to analyse the fragment sizes.

 

What quality controls are in place to verify the production of cfDNA HDx Reference Standards?

The following tests are performed on each batch to ensure the consistency and accuracy of the Reference Standards:

TestsTest Method

Fragmentation Size

D1000 DNA ScreenTape assay

Allelic Frequency

Droplet Digital™ PCR

Quantification

Qubit dsDNA BR Assay (post-fragmentation)

 

Can I use cfDNA HDx Reference Standards on my platform?

cfDNA HDx Reference Standards are adaptable to most platforms. Our partners have tested the utility of these cfDNA HDx Reference Standards using qPCR, digital PCR and amplicon based NGS platforms. Please contact us at technical@horizondiscovery.com if you have any further questions.

 

Can we use HDx Reference Standards as a spike-in reference?

Yes. The application of cfDNA HDx Reference Standards is to support validation studies. They can also be used as a spike-in reference to assess the efficiency of cfDNA extraction workflow for cfDNA analysis.

cfDNA HDx Reference Standards are used on its own. However if users intend to use the reference standard as a spike-in reference, we recommend that the reference standards are spiked into healthy donor plasma. Users are encouraged to run cfDNA HDx Reference Standards as provided to assess how the reference standard perform on their workflow and platform prior carrying out the spike-in experiment. Users are advised to define the amount of reference standards to spike into healthy donor plasma. The allelic frequencies called in spiked-in reference are lower and more variable compared to the allelic frequencies called with the provided reference standards. This is due to the presence of wild-type cfDNA in the normal donor plasma.

 

Will I see competition between probes when using a multiplex assay?

The Multiplex I cfDNA Reference Standard Set contains two mutations at very close proximity: NRAS Q61K and NRAS A59T. Competition between the probes can be observed when analysing Multiplex I for the NRAS A59T mutation, Eg. an extra population can be observed in 2D plot ddPCR analysis mode probably representing non-specific amplification of the NRAS Q61K mutation.

 

What does it mean if my results look different to the product Insert?

The product insert provides the expected allelic frequencies and Horizon’s observed allelic frequencies using an in-house ddPCR assay. Any observational differences will be due to the variability in assay design, platforms and workflows. Please contact us at technical@horizondiscovery.com if you have any questions.

The limit of detection can be assessed by comparing the expected and observed allelic frequencies at different ranges (i.e. 5%, 1%, 0.1%). A significant difference of allelic frequencies measured compared to the expected allelic frequencies suggests a low sensitivity and gives an idea on the limit of detection of the assay. The 100% Wild Type cfDNA Reference Standard is a useful negative control for evaluating assay contamination and false positive rates.

Please refer to application note for utility of Multiplex I cfDNA reference standard.

 

Are there any additional mutations found in the cfDNA Reference Standards?

For information on endogenous mutations in these reference standards, you may download whole-exome sequencing data of the parental cell lines for each product from here or download link provided on respective product page.

 

Is it possible to order custom versions of the cfDNA Reference Standard?

Horizon is pleased to accept custom cfDNA reference standards requests. Timeline and pricing of custom request is decided on a case-by-case basis depending on panel complexity. Please contact us by email at info@horizondiscovery.com.

 

Please find below list of publications demonstrating the utility of cfDNA HDx Reference Standards

  • Multiplex I cfDNA Reference Standard Set application note
  • Poster in collaboration with CareDx on application of cfDNA in solid organ transplantation

 

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