TrueMethyl® oxBS-Seq Module

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TrueMethyl^® oxBS-Seq

DNA methylation plays a critical role in many regulatory processes in a cell. The analysis of genome-wide epigenetic changes including DNA methylation and hydroxymethylation has become a subject of serious research for many biological and disease-associated studies.

With the integration of TrueMethyl® oxBS-Seq chemistry, users can now gain new insights into epigenetic modifications with the ability to interrogate both 5mC and 5hmC in a single protocol. Methyl-Seq kits work hand-in-hand with Tecan’s DNA-Seq and RNA-Seq kits to interrogate all aspects of epigenetic regulation. Streamlined, reliable, user-friendly protocols that deliver results.

Selection guide

TrueMethyl oxBS-Seq with integrated oxidative bisulfite conversion

As the exclusive partner of Cambridge Epigenetix, Tecan is the source for the breakthrough approach of oxidative bisulfite conversion. 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® Ultralow Methyl-Seq and Ovation® RRBS Methyl-Seq kits, the result is accurate, low-cost whole genome or RRBS sequencing.

For more in-depth information on how this technology can be applied to mapping hydroxymethylation patterns in neurobiological systems, please see the Spotlight on Science featuring Dr. Jonathan Mill from the University of Exeter Medical School.

Features of the TrueMethyl oxBS-Seq when combined with Tecan's library preparation kits include:

DimerFree library preparation
Integrated oxidative bisulfite conversion
Uniform whole genome coverage with Ovation Ultralow Methyl-Seq kit
Reproducible RRBS enrichment with Ovation RRBS Methyl-Seq kit
Detection of 5mC and 5hmC with the same workflow
Directional libraries
All-in-one: From DNA to sequenceable library in a single kit

Applications

Methyl-Seq
Reduced Representation Bisulfite Sequencing (RRBS)
Bisulfite sequencing
Oxidative bisulfite sequencing
DNA Methylation arrays

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

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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.

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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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Ultralow Methyl-Seq Library Kit with TrueMethyl oxBS

The Ultralow Methyl-Seq Library kits provide a simple, fast and scalable solution for producing libraries used in conjunction with bisulfite sequencing to analyze DNA methylation.

With input amounts down to 10 ng the kit enables methylation studies for a broad range of sample types and can be completed in approximately 9 hours.

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Obtain high quality results regardless of input.

Libraries were generated from 100 ng, 10 ng and 1 ng of human genomic DNA. Bioanalyzer analysis indicates no adaptor artifacts (grey box) regardless of input and without the need for adaptor dilution during library construction.

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Directional libraries, simplified data analysis

Nucleotide Distributions of Forward (A) and Reverse (B) Reads. Maintaining the directional orientation of the original genomic DNA reduces the computational burden by a factor of two over non-directional libraries during Bismark data analysis. The forward read is always the C-to-T converted (original genomic) strand, while the reverse read is the G-to-A reverse complement.

Ovation RRBS Methyl-Seq kit with TrueMethyl oxBS

The Ovation RRBS Methyl-Seq kit provides a complete 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 without the need to sequence the entire genome.
Suitable sample input is human genomic DNA from a broad range of cell and tissue types.

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With RRBS there is no need to sequence the entire genome.

Top: Diagram of the Ovation RRBS Methyl-Seq kit final library structure. The location of the diversity bases and molecular tag (N6) are shown. Bottom: Graphical representation of how unique molecules are identified based on the read alignment and molecular tag (N6) sequence.

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Obtain results that are highly reproducible and concordant.

Concordance in methylation levels for CpG’s covered at 20x or greater depth between technical replicates using 25 ng of IMR90 gDNA (Left) and between Ovation RRBS Methyl-Seq System versus Whole Genome Bisulfite Sequencing (WGBS, Right).

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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

FAQ

Input recommendations

Can I use FFPE or other degraded DNA as input into TrueMethyl oxBS Module?

Yes. When working with FFPE or degraded DNA, we recommend inputs of 500 ng-1 µg.

General workflow

Can I prepare MBBS1 and MBBS2 in advance/prepare extra solution?

It is recommended to prepare MBBS1 and MBBS2 fresh on the day of use. If reagent is prepared in advance or if excess reagent is prepared, store at 4 °C and use within one week. Discard after 1 week.

I don’t have access to a Covaris instrument. Can I use alternative fragmentation methods?

We have evaluated only Covaris fragmented DNA during the development of the Ultralow Methyl-Seq library prep kit. Other mechanical means of fragmentation, such as hydrodynamic shearing or nebulization, may be suitable.

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.

Does Tecan provide reagents for performing the fragmentation step of the protocol?

Tecan does not provide the reagents used in the fragmentation steps. We suggest the Covaris instrument be utilized for DNA fragmentation, as suggested in the “materials” section of the User Guide.

Literature

Type

Title

User Guide

Ovation RRBS Methyl-Seq System

M01394 V7

User Guide

TrueMethyl oxBS Module

M01481 V3

User Guide

Ultralow Methyl-Seq with TrueMethyl oxBS Module

M01512 V2

Product Sheet

Methyl-Seq Solutions

401213 V1

About Tecan

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TrueMethyl^® oxBS-Seq Module