Ovation® RRBS Methyl-Seq System
The Ovation® RRBS Methyl-Seq System provides a simple, fast and scalable solution for producing Methyl-Seq libraries using the reduced representation bisulfite sequencing approach to analyze DNA methylation.
Reduced Representation Bisulfite Sequencing or RRBS is a method that delivers genome-wide quantitative DNA methylation information at single base resolution.
Suitable sample input is human genomic DNA from a broad range of cell and tissue types.
Get more informative reads
- Built in sequence diversity eliminates the need for PhiX
- Diversity bases improve quality and produce more informative reads with improved quality
- Integrated molecular tag (N6) enables removal of non-unique reads from the dataset
Obtain results that are highly reproducible and concordant
- Methylation data concordant with whole genome bisulfite sequencing data
Complete the workflow within a day
- Direct adaptor ligation with no need for A-tailing and end repair
- Convenient protocol
- Bisulfite sequencing
- Reduced Representation Bisulfite Sequencing (RRBS)
|Compatible Platform||Illumina HiSeq, MiSeq, NextSeq|
|Starting Material||purified genomic DNA|
|Input Amounts||100 ng|
We recommend using high quality DNA with an A260:A280 ratio of 1.7-2.0. Although not recommended, it is possible to generate WGBS libraries from less than 100 ng of gDNA, or from degraded gDNA, such as DNA etracted from formalin fied, paraffin embedded (FFPE) specimens. If you choose to proceed with such samples, Library Amplification will require additional cycles of PCR. Please see the User Guide for guidelines, or follow the optional real-time PCR protocol in Appendi B.
NuGEN does not provide the reagents used in the bisulfite conversion steps. We recommend using the QIAGEN EpiTect Fast DNA Bisulfite Kit (QIAGEN Cat. #59824). Other commercial bisulfite conversion kits may be suitable as well, but these have not been validated.
Can I modify the number of PCR amplification cycles recommended by the Ovation RRBS Methyl-Seq System 1–16 workflow when using different DNA input amounts?
Real-time PCR can be used to determine the appropriate number of PCR cycles. Please see Appendi B in the User Guide for a detailed protocol. For more information, contact NuGEN Technical Support.
Can I combine the barcoded libraries prior to the PCR amplification step?
This is not recommended. The stoichiometry of barcoded libraries may be adversely affected by this modification to the workflow. We suggest that the libraries be amplified and quantitated independently before being pooled for use on the sequencer.
RNAClean P beads are certified to be RNase and DNase free. We have tested both RNAClean P and AMPure P beads in our kits and observe no difference in performance between products.
What magnetic separation devices do you recommend for the SPRI bead purifications?
Due to the large number of commercially available magnets, we do not have a comprehensive list of compatible products. However, many magnets are compatible. As long as the magnet is strong enough to clear the solution of magnetic beads, it can be applied to the system. We have the following guidelines for selecting a magnetic separation device:
- Use a magnet designed for 0.2 mL tubes (PCR tubes), tube strips, or plates. Compared to magnets that are designed for 1.5 mL tubes, these minimize loss that can occur when samples are transferred from one tube to another.
- Prior to purchasing, check the manufacturer’s specifications for minimum and maimum volumes that can be effectively treated.
- Test the magnet with a mock purification to ensure the magnet will effectively clear the solution under the conditions in the NuGEN workflow. This is also helpful to gain familiarity with the purification workflow.
How can I ensure maimum recovery of sample from the SPRI bead purification?
- Allow the SPRI beads to reach room temperature before use; cold beads result in lower yields.
- Ensure that the beads are fully resuspended in solution before adding to the sample.
- Always use fresh ethanol during the washing steps. When preparing the ethanol, measure out the ethanol and water separately to ensure the desired ethanol concentration is obtained.
- Mi the bead suspension and sample thoroughly to ensure maimum binding of the samples to the beads.
Please refer to section V.K. of the User Guide for guidelines on quantitative and qualitative assessment. We recommend using a qPCR based-method such as the in combination with the Agilent Bioanalyzer or Tapestation for the most accurate quantification.
What is the epected yield of the amplified DNA library using the Ovation RRBS Methyl-Seq System 1–16?
The epected yield is at least 200 ng, depending on the quality and quantity of the genomic DNA and the number of PCR cycles employed. This amount is in ecess of the amount of DNA required for cluster generation.
How many bases do the Ovation RRBS Methyl-Seq System adaptors add to the library?
The adaptors add 145 bp to the library.
Ovation RRBS Methyl-Seq Systems libraries are compatible with Illumina sequencing platforms. Custom sequencing primers may not be supported on all platforms. Please see the recommendations for your specific sequencer.
How much material should I load into the sequencer?
Please follow manufacturer’s recommendations for library QC, quantitation, balancing and loading of the amplified library on the sequencer.
What kind of sequencing primers can I use with your libraries?
The design of the Ovation RRBS Methyl-Seq System 1–16 requires the use of a custom Read 1 sequencing primer, MetSeq Primer 1, which is included in this kit at a concentration of 25 μM. The standard primers provided in the Illumina sequencing kit are sufficient for Read 2 and for sequencing the barcodes (Inde Read). The Standard Read 1 Primer is also required when using Phi or other libraries to increase base compleity.
What kind of error correction is used to minimize the impact of sequencing errors in the barcodes?
Each barcode is a minimum edit distance of 3 from any other barcode. This means that a minimum of three edits (replacement, insertion, or deletion) must occur before one barcode becomes a different barcode. For further details on the barcode design strategy, please refer to Faircloth BC, Glenn TC (2012), Not All Sequence Tags Are Created Equal: Designing and Validating Sequence Identification Tags Robust to Indels. PLoS ONE 7(8): e42543. doi:10.1371/journal.pone.0042543.
Can the Ovation RRBS Methyl-Seq System 1–16 be used with paired-end sequencing?
Yes, it can be used for both single end and paired-end sequencing.
Yes, the libraries are directional due to the way our library system is designed and the nature of bisulfite conversion. The forward sequencing reads will correspond to a bisulfite-converted version of either the original top or the original bottom strand (the C-to-T reads) and the reverse sequencing reads will correspond to the complement of the original top or the complement of the original bottom strand (the G-to-A reads). In contrast, a non-directional bisulfite converted library will have all four possible strands in the forward read (original top, original bottom, complement of original top and complement of original bottom).
What analysis software can be used for aligning, methylation calling, and visualization of your bisulfite sequencing data?
The number of analysis strategies and software tools for methylation-based sequencing studies is growing rapidly. The ideal analysis workflow for a given eperiment depends on many variables, including the type of eperiment and the goals of the study. Currently, NuGEN scientists use Bismark for aligning and determining methylation status. This program utilizes the Bowtie aligner (www.bioinformatics.bbsrc.ac.uk/projects/bismark/). The Broad IGV genome browser can be used to visualize the results of Bismark (http://www.broadinstitute.org/igv/).
How can I measure the efficiency of bisulfite conversion?
DNA material that is known to be unmethylated, such as lambda DNA, can be used to measure the efficiency of C-to-U conversion in the bisulfite conversion kit. This control DNA is not included with the Ovation RRBS Methyl-Seq System 1–16.
For research use only. Not for use in diagnostic procedures.