We have compiled on this page the answers to the questions that scientists ask most frequently about TrueMethyl and oxidative bisulfite (oxBS) technology. If you have additional questions please contact us.
EPIGENETICS AND DNA MODIFICATIONS
TRUEMETHYL OXIDATIVE BISULFITE TECHNOLOGY
- What are the advantages of TrueMethyl?
- How does TrueMethyl work?
- What is included in the TrueMethyl kit?
- Do you provide a DNA modification profiling service?
- Are there any oxBS-Seq publications?
- What is the DNA input requirement per sample?
- Does the oxidation step cause DNA damage?
- Can I use the Cambridge Epigenetix oxidant in combination with my traditional bisulfite method?
- Do you sell sub-components of the TrueMethyl kit?
- How long are the TrueMethyl processed BS/oxBS samples stable for?
COMPATIBILITY WITH OTHER APPLICATIONS AND PLATFORMS
- Which platforms are compatible with TrueMethyl?
- Which applications are compatible with TrueMethyl?
- Which Next Generation Sequencing library preparation methods are compatible with TrueMethyl?
SHIPMENT & KIT STORAGE
- How is the TrueMethyl kit shipped?
- How do I store the kit appropriately?
- What is the shelf life of the TrueMethyl kit?
EPIGENETICS AND DNA MODIFICATIONS
It's been known for some time that cytosine can be modified into 5-methylcytosine (5mC), an epigenetic mark that regulates gene expression and is involved in several aspects of human health and disease. More recently scientists have discovered that 5mC can be oxidised in vivo into other cytosine variants, namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxycytosine (5caC). These cytosine variants are gaining importance in several fields of biology including cancer, stem cells and neuroscience. They are stable marks, are present at high levels in certain human cell types, have distinct localisation and binding partners from each other and 5mC, and have been linked with reactivation of gene expression.
The discovery of hydroxymethylation has proven a challenge for researchers since established techniques have significant limitations: antibody-based approaches suffer from poor specificity/selectivity and enrichment or pull-down methods of this type have low resolution and are not quantitative. Third generation sequencing techniques are still in their infancy and produce DNA methylation read-outs that are difficult to decipher. Importantly, traditional bisulfite (BS) sequencing, the gold standard in DNA methylation analysis, fails to discriminate between 5hmC and 5mC, and yields a result that combines the two in an unknown proportion.
To overcome these issues we have developed TrueMethyl, an innovative solution based on oxidative bisulfite (oxBS) technology that enables researchers to distinguish DNA methylation (5mC) and hydroxymethylation (5hmC), and quantify them at single base resolution.
Please visit the resources section of our website to learn more about the importance of DNA modifications and the TrueMethyl technology.
TRUEMETHYL OXIDATIVE BISULFITE TECHNOLOGY
TrueMethyl is the only method that allows you to accurately and quantitatively measure true levels of 5-methylcytosine (5mC) at single base resolution.
In addition to 5mC, with TrueMethyl you can accurately and quantitatively measure 5-hydroxymethylcytosine (5hmC) at single base resolution.
TrueMethyl is a robust and reproducible method that is compatible with common workflows, including next generation sequencing, Illumina Infinium HumanMethylation450 BeadChip (450K array), and locus specific approaches. Please refer to our protocols page for details on specific applications.
TrueMethyl is based on oxidative bisulfite (oxBS) technology, which uses a selective chemical oxidation to accurately detect and distinguish between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) at single base resolution.
Firstly, chemical oxidation using our CEGX proprietary oxidant, converts 5hmC into 5-formylcytosine (5fC). A specifically formulated TrueMethyl bisulfite treatment then converts 5fC and C to uracil, leaving only 5mC to be detected by sequencing. This gives quantitative measurement of true levels of 5mC at single-base resolution.
If you wish to profile 5hmC, you can do so by running a parallel bisulfite sequencing (BS-Seq) run with TrueMethyl, omitting the oxidation step, which yields 5mC+5hmC. Specific 5hmC bases can be identified by subtracting the TrueMethyl oxBS dataset from the TrueMethyl BS dataset.
It is important to compare oxBS and BS datasets originating from the same experiment to obtain accurate and quantitative results. We advise against comparing TrueMethyl oxidative bisulfite datasets to pre-existing (non-TrueMethyl) bisulfite datasets to obtain 5hmC profiles. Such datasets are not comparable with each other due to use of different reagents and experimental conditions.
TrueMethyl is a full product solution that contains all of the reagents you need to successfully convert your DNA samples for the subsequent measurement of true DNA (hydroxy)methylation. The TrueMethyl kit includes:
1. A stringent purification step, essential to eliminate contaminants that can cross-react with reagents in the protocol.
2. An enhanced oxidation formulation that converts 5hmC to 5fC.
3. An optimised bisulfite conversion reagent specifically formulated to achieve 5fC to uracil conversion.
The TrueMethyl Seq Kit also includes a PCR amplification module that includes the TrueMethyl DNA Polymerase, a mutant polymerase that has been engineered to be resistant to uracil stalling and therefore is ideal for use in the amplification of TrueMethyl-converted libraries.
These components have been designed and optimised to work well together and results cannot be guaranteed if other components or reagents are substituted in place of validated TrueMethyl reagents.
Our TrueMethyl kits include spike-in controls that we have developed to allow you to monitor the success of your experiment. The TrueMethyl spike-in controls are:
1. A Digestion Control for qualitative assessment of 5hmC oxidation and bisulfite conversion prior to committing samples to sequencing (included in the TrueMethyl Array and TrueMethyl Seq Kits)
2. A Sequencing Control (provided in the TrueMethyl Seq Kit), consisting of a pool of 6 oligonucleotide duplexes containing C, 5mC, 5hmC and 5fC at known positions and density and can be interrogated after sequencing using bsExpress (link here) to give a quantitative assessment of the efficiency of conversion for every TrueMethyl experiment.
The TrueMethyl spike-in controls are HPLC purified synthetic oligonucleotide duplexes. These controls are of superior quality and do not suffer from the same technical limitations as PCR generated controls. The exact composition of PCR generated controls cannot be guaranteed due to the presence of contaminating dCTP in some dhmCTP preparations and we do not recommend use of homemade PCR generated controls for bisulfite based experiments for this reason.
Please refer to the user guide for further details.
We currently don’t offer a DNA modification profiling service. But we are partnering with service providers to do so. If you are interested in learning more, please contact us at firstname.lastname@example.org.
Yes. We have compiled a list of high impact publications utilising oxidative bisulfite to study DNA (hydroxy)methylation in various fields including cancer biology, development, stem cell research, and neuroscience. Please check out the publications page of our website.
Currently, the validated input masses are:
1. For TrueMethyl Seq – 50 - 500 ng of adapted NGS library for the oxBS sample and 50 - 500 ng of adapted NGS library for the BS-only sample (for the complete protocol refer to our TrueMethyl Seq user guide). The TrueMethyl Seq Kit has been designed and optimised for processing NGS libraries through the TrueMethyl workflow.
2. For TrueMethyl Array – 1 μg is the recommended starting quantity to produce sufficient material for the 450k methylation array. This is subsequently split between oxBS and BS reactions (for the complete protocol refer to our TrueMethyl Array user guide). The TrueMethyl Array Kit has been designed and optimised for processing HMW genomic DNA through the TrueMethyl workflow and is compatible with input masses as low as 50ng for such post-BS applications as pyrosequencing and loci-specific amplicon sequencing.
No evidence exists for non-specific DNA damage arising from the chemical treatment of DNA specific to the oxidation step. The recovery of DNA from the oxBS-Seq workflow is comparable to that from BS-Seq only. The size of the fragments obtained with TrueMethyl is slightly shorter compared to those obtained with standard bisulfite methods, due to the longer bisulfite reaction that is necessary to convert 5fC to Uracil.
All components of the TrueMethyl kit are optimised to work together for the best results. Enhanced proprietary chemistry and optimised conditions for oxidative bisulfite will guarantee successful quantification of 5mC and 5hmC.
The TrueMethyl bisulfite conversion reagents and incubation conditions are specifically formulated for optimised conversion of 5fC to uracil, unlike other methods that are not designed to eliminate endogenous 5hmC. We therefore advise against substituting individual components of the TrueMethyl workflow that have not been validated by Cambridge Epigenetix .
Moreover, it is important to compare oxBS and BS datasets generated with the TrueMethyl protocol when quantifying 5hmC. Only comparing datasets obtained with the same chemistry and protocol will yield accurate, quantitative data that you can trust.
No. TrueMethyl is a full product solution that contains all you need to successfully convert your DNA samples, including essential purification reagents, an enhanced oxidation formulation, a specifically formulated bisulfite reagent, a uracil-tolerant PCR module, and two sets of spike-in controls.
These components have been designed and optimised to work well together and results cannot be guaranteed otherwise. If you follow the TrueMethyl workflow according to the user guide and using the components provided in the kit, results are guaranteed. If a component of your kit is missing please contact us at email@example.com.
TrueMethyl converted samples may be stored like any other bisulfite converted sample. For long-term storage we recommend storage at -20C.
COMPATIBILITY WITH OTHER APPLICATIONS AND PLATFORMS
TrueMethyl is compatible with a variety of common platforms, including next generation sequencing, Illumina Infinium HumanMethylation450 BeadChip (450K array), and targeted assays. For next generation sequencing please refer to our TrueMethyl Seq user guide; for 450K array please refer to our TrueMethyl Array user guide.
Please sign up for our newsletter and refer to our protocols page for details and updates. If you have a specific platform that you would like to use in combination with TrueMethyl please contact us at firstname.lastname@example.org.
The Cambridge Epigenetix R&D team is currently developing new application note protocols to enable use of TrueMethyl with other popular platforms. Please sign up for our newsletter and refer to our protocols page for details and updates. If you have a specific platform that you would like to use in combination with TrueMethyl please contact us at email@example.com.
TrueMethyl is compatible with a variety of applications. Please refer to our TrueMethyl user guides to select the appropriate kit for your chosen application or contact firstname.lastname@example.org for further information.
Please note that some deviations from the protocol described in the TrueMethyl User Guide may be necessary for particular applications. In particular, it is important to be aware of input/output masses for these techniques and the TrueMethyl workflow.
The Cambridge Epigenetix R&D team is currently developing new application note protocols to enable use of TrueMethyl with other popular applications. Please sign up for our newsletter and refer to our protocols page for details and updates. If you have questions regarding a specific application please contact us at email@example.com.
For pre-bisulfite library preparation, please follow the guidelines below:
1. The sequencing adapters MUST BE METHYLATED
2. The recovered library mass must be compatible with TrueMethyl Seq DNA input requirements.
Please check with vendors for specifications.
Although in principle TrueMethyl is compatible with any library preparation method, the current workflow has only been validated for pre-bisulfite library construction. If you are interested in combining TrueMethyl with a post-bisulfite library preparation method, contact us at firstname.lastname@example.org for advice.
We have developed two bioinformatic tools, bsExpress and cegxQC, to enable fast and simple analysis of our TrueMethyl spike-in sequencing controls. bsExpress will enable you to assess the quality of your data by determining the various conversion rates of your oxidative-bisulfite and bisulfite-only treated samples. For complete biological analysis of your data you should consult with your bioinformatician. We are working to develop additional bioinformatic tools to help you to analyse TrueMethyl datasets and can also provide bioinformatics guidance. Please visit our website and subscribe to our newsletter for updates. If you have specific questions you can also contact us at email@example.com.
SHIPMENT & KIT STORAGE
The TrueMethyl Kit is shipped as two separate boxes:
1. oxBox 1 is shipped at ambient temperature (15 – 25°C).
2. oxBox 2 is shipped frozen (-20 °C).
IMPORTANT: The oxidant solution is sensitive to carbon dioxide exposure. Under no circumstance should the oxidant come into contact with CO2 or dry ice otherwise performance will be significantly impaired. The oxidant is shipped in oxBox 2 on frozen ice packs and not dry ice for this reason.
1. The Magnetic Bead Solution, Binding Buffer 1 and Binding Buffer 2 should be removed from oxBox1 and stored in the fridge at 2-8°C. DO NOT FREEZE these components; they should only be stored in the fridge at 2-8°C. Short-term storage of these components at room temperature does not affect their performance.
2. All other buffers and the bisulfite reagent should be stored at room temperature and are stable for 18 months from the date of manufacturing under these conditions. The kit expiry date is indicated on oxBox1.
3. Dissolved Bisulfite Mix should be used immediately and then disposed of appropriately. It must not be stored and reused. For optimal use of the kit components, please plan your experiments to ensure samples are processed in appropriately sized batches. For the TrueMethyl Seq Kit, one aliquot of dissolved bisulfite reagent is sufficient for 4 bisulfite reactions (2 x oxBS plus 2 x BS). For the TrueMethyl Array Kit, one aliquot of dissolved bisulfite reagent is sufficient for 24 bisulfite reactions (12 x oxBS plus 12 x BS).
OxBox 2 -
1. oxBox 2 and its contents should be stored at -20°C.
The TrueMethyl guaranteed shelf life is currently 18 months from the date of manufacturing. The expiry date is indicated on the kit packaging.