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Ovation® RRBS Methyl-Seq with TrueMethyl® oxBS

Fast and scalable solution for producing Methyl-Seq libraries using the Reduced Representation Bisulfite Sequencing approach to analyze DNA methylation.
Overview
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Tab 01 / Overview
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What is Reduced Representation Bisulfite Sequencing (RRBS)?

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. 

 

Selection guide

The Ovation RRBS Methyl-Seq library preparation kit

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.


Features of the Ovation RRBS Methyl-Seq Kit:

  • DimerFree™ library preparation
  • Integrated oxidative bisulfite conversion (TrueMethyl)
  • Uniform whole genome coverage
  • Reproducible RRBS enrichment
  • PhiX-free sequencing with diversity adaptors
  • Detection of 5mC and 5hmC with the same workflow
  • Directional libraries
  • All-in-one: From DNA to sequenceable library in a single kit

Applications of RRBS:

  • Investigating epigenetic changes related to carcinogenesis
  • Investigating genetic diseases such as Down syndrome and Huntington in neuroscience
  • Studying the mechanism of immune systems
  • Studying developmental biology by monitoring methylated regions




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Tab 02 / Highlights
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Get more informative methyl-seq 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

 

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.

No detectable adaptor dimers.

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.

A higher % of informative reads

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.

Obtain results that are highly reproducible and concordant

  • DNA methylation data concordant with whole-genome bisulfite sequencing data

 

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).

Reproducibility of libraries from Ovation Library Systems

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.

Sequencing alignment metrics with MAQC samples

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.

Complete the methyl-seq workflow within a day

  • Direct adaptor ligation with no need for A-tailing and end repair
  • Convenient protocol

Compatible with the TrueMethyl oxBS Module

The TrueMethyl oxBS Module contains the necessary reagents for quantification of the true level of cytosine methylation (5mC) and hydroxymethylation (5hmC).

  • Cost-effective, optimized analysis of both 5mC and 5hmC
  • Sensitive methylation detection from as little as 100 ng DNA
  • Streamlined protocol enables processing of 32 oxBS reactions and 32 BS-only reactions in <6 hours
  • Simple data analysis workflow and expert support
Show more

Achieve complete measurements of methylation with oxBS.

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.

TrueMethyl oxidative bisulfite conversion enables accurate measurements of methylation.

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.




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Tab 03 / Specifications
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Specifications

Specification

Specification/Description

Compatible Platform

Compatible Platform

Illumina HiSeq, MiSeq, NextSeq, MiniSeq, NovaSeq

Starting Material

Starting Material

Purified genomic DNA

Input Amounts

Input Amounts

100 ng




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Tab 04 / Faq
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FAQ

Getting started

Does Tecan provide reagents for performing the bisulfite conversion step of the protocol?

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.

Input recommendations

Can I use less than 100 ng of input DNA into the Ovation RRBS Methyl-Seq System?

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.

General workflow

Can I use restriction enzymes other than, or in addition to, MspI?

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.

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 quantified independently before being pooled for use on the sequencer.

Why doesn’t my Library Amplification qPCR plot resemble the example in the user guide?

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.

SPRI bead purifications

What is the difference between RNAClean XP and AMPure XP SPRI beads? Can both be used interchangeably?

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.

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:

  1. 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.
  2. Prior to purchasing, check the manufacturer’s specifications for minimum and maximum volumes that can be effectively treated.
  3. Test the magnet with a mock purification to ensure the magnet will effectively clear the solution under the conditions in the Tecan workflow. This is also helpful to gain familiarity with the purification workflow.

How can I ensure maximum recovery of sample from the SPRI bead purification?

  1. Allow the SPRI beads to reach room temperature before use; cold beads result in lower yields.
  2. Ensure that the beads are fully resuspended in solution before adding to the sample.
  3. 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.
  4. Mix the bead suspension and sample thoroughly to ensure maximum binding of the samples to the beads.

Library quantification and qualification

How do I measure my final library yield? Can I use an Agilent Bioanalyzer to evaluate the product?

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.

What is the expected yield of the amplified DNA library using the Ovation RRBS Methyl-Seq with TrueMethyl oxBS?

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.

How many bases do the Ovation RRBS Methyl-Seq adaptors add to the library?

The adaptors add 145 bp to the library.

Sequencing recommendations

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 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.

Can the Ovation RRBS Methyl-Seq System 1–16 be used with paired-end sequencing?

Yes, it can be used for both single- and paired-end sequencing.

How much material should I load into the sequencer?

Please follow manufacturer’s recommendations for library QC, quantification, balancing and loading of the amplified library on the sequencer.

Is the Ovation RRBS Methyl-Seq System compatible with all Illumina sequencing platforms?

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.

Data analysis

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.

Are the libraries directional?

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 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

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.




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Tab 05 / Literature
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Literature
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