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Fast and scalable solution for producing Methyl-Seq libraries using the Reduced Representation Bisulfite Sequencing approach to analyze DNA methylation.
Conventional bisulfite sequencing techniques are not able to properly sequence highly repetitive methylated regions. RRBS is a genome-wide DNA methylation analysis, which only sequences a reduced, representative sample of whole genome, reduces the cost of whole-genome sequencing.
The Ovation RRBS Methyl-Seq library preparation kit delivers genome-wide quantitative DNA methylation information at single base resolution. This kit is a simple, fast, and scalable solution for producing Methyl-Seq libraries using the RRBS approach to analyze DNA methylation. Our Ovation RRBS Methyl-Seq kit incorporates diversity adaptors to eliminate the need for PhiX and thus save on wasted sequencing costs. Suitable sample input is human genomic DNA from a broad range of cell and tissue types.
As the exclusive partner of Cambridge Epigenetix, Tecan's Methyl-Seq kits are the only ones that contain the breakthrough TrueMethyl oxidative bisulfite conversion technology. This method enables the most accurate 5-methylcytosine (5mC) identification for NGS, and the interrogation of 5-hydroxymethylcytosine (5hmC), a modified base that is not assayed by traditional bisulfite conversion approaches. When used in conjunction with the Ovation RRBS Methyl-Seq kit, the result is accurate, low-cost RRBS sequencing.
Diagram of the Ovation RRBS Methyl-Seq kit final library structure. The location of the diversity bases and molecular tag (N6) are shown. Graphical representation of how unique molecules are identified based on the read alignment and molecular tag (N6) sequence.
1 ng gDNA created from Ultralow System V2 vs. Competitors X and Y analyzed on Agilent Bioanalyzer. Adaptor dimers are highlighted in the grey box.
Ovation® Ultralow System V2 results in the lowest amount of adaptor dimers produced, and 50% more informative reads than the competition at a 10 pg input.
Concordance in methylation levels for CpG’s covered at 20X or greater depth between technical replicates (left) and between Ovation RRBS Methyl-Seq Kit versus Whole Genome Bisulfite Sequencing (WGBS, right).
Concordance plots demonstrating reproducibility of the integrated workflow using Ovation® RNA-Seq System V2 with Tecan’s Ovation® Rapid Library System with differing amounts of SPIA cDNA input to the library construction workflow. RPKM plots average R ≥ 0.9 for different library inputs.
Data was generated using 2 ng of Total RNA from either Human Brain (MAQC B) or Universal Human Reference (MAQC A) input to the Ovation® RNA-Seq System V2 followed by library construction using the Encore NGS Library System I. The resulting libraries were sequenced on the Illumina Genome Analyzer IIx with 40 bp single-read sequencing.
The TrueMethyl oxBS Module contains the necessary reagents for quantification of the true level of cytosine methylation (5mC) and hydroxymethylation (5hmC).
The schematic (Left) shows classic bisulfite conversion, which creates a library that detects both 5mC and 5hmC. Processing with the oxidation of 5hmC (Right) generates a bisulfite-convertible base that leads to detection of only 5mC. Differences between the libraries can then be used to deduce the sites of 5hmC modifications.
A) Standard bisulfite conversion cannot distinguish between 5mC and 5hmC, resulting in a single readout. B) TrueMethyl oxidative bisulfite conversion provides an accurate methylation profile of each.
Illumina HiSeq, MiSeq, NextSeq, MiniSeq, NovaSeq
Purified genomic DNA
Yes, the bisulfite conversion reagents are included with the purchase of product part number 0553-32. This bundle includes the Ovation RRBS Methyl-Seq core kit (Part No. 0353) and the TrueMethyl oxBS module (Part No. 0414). The QIAGEN EpiTect Fast DNA Bisulfite Kit (QIAGEN Cat. #59824) is a suitable substitute. Other commercially available bisulfite conversion kits may be suitable as well, but these have not been validated.
The Ovation RRBS Methyl-Seq system with the TrueMethyl oxBS workflow has not been tested for inputs less than 100 ng. Although not recommended, the Ovation RRBS Methyl-Seq System core kit may be used with third-party bisulfite conversion kits for less than 100 ng inputs. See Appendix B. for recommendations on working with low-input samples.
Yes, however the restriction site must leave a similar 3’-CG overhang in order for the ligation to be effective. Please contact Tecan NGS technical support for further advice on integrating other enzymes into this protocol.
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 quantified independently before being pooled for use on the sequencer.
Certain real-time PCR instruments may display unexpected results, such as the example in the FAQ section of the User Guide. Ensure that your plot is set to display Rn. vs. Cycle, not deltaRn vs. Cycle, and that the y-axis is set to a log scale.
RNAClean XP beads are certified to be RNase and DNase free. We have tested both RNAClean XP and AMPure XP beads in our kits and observe no difference in performance between products.
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:
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 Fragment Analyzer for the most accurate quantification.
The expected 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 excess of the amount of DNA required for sequencing.
The adaptors add 145 bp to the library.
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 100 μM. The standard primers provided in the Illumina sequencing kit are sufficient for Read 2 and for sequencing the barcodes (Index Read). The Standard Read 1 Primer is also required when using PhiX or other libraries to increase base complexity.
Yes, it can be used for both single- and paired-end sequencing.
Please follow manufacturer’s recommendations for library QC, quantification, balancing and loading of the amplified library on the sequencer.
Ovation RRBS Methyl-Seq Systems libraries are compatible with Illumina sequencing platforms. Please see the recommendations on using custom sequencing primers for your specific sequencer.
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.
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).
The number of analysis strategies and software tools for methylation-based sequencing studies is growing rapidly. The ideal analysis workflow for a given experiment depends on many variables, including the type of experiment and the goals of the study. Currently, Tecan 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/). Data analysis recommendations can be found here: https://github.com/tecangenomics/NuMetWG
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.
Unique TrueMethyl oxBS technology enables the most accurate 5mC identification...
Simple library preparation kit provides a fast and scalable solution for producing...
For research use only. Not for use in diagnostic procedures.