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Ultralow Methyl-Seq with TrueMethyl® oxBS

Fast and scalable solution for producing Methyl-seq libraries for Whole genome methylation profiling.
Overview
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Tab 01 / Overview
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Ultralow Methyl-Seq: from DNA to sequenceable Methyl-seq library in a single kit

The analysis of DNA methylation as a genome-wide epigenomic alteration has become a subject of research in many areas of life science. Applying NGS to do whole-genome bisulfite sequencing (WGBS) is a gold standard technique for a complete and quantitative analysis of cytosine methylation throughout the genome.

 

Selection guide

The Ultralow Methyl-Seq library preparation kit provides a simple, fast and scalable solution for producing libraries used in conjunction with bisulfite sequencing to analyze DNA methylation. With input levels as low as 10 ng, the kit enables methylation studies for a broad range of sample types and can be completed in approximately 9 hours.

As an exclusive partner of Cambridge Epigenetix, Tecan is the source for the breakthrough approach of oxidative bisulfite conversion. TrueMethyl oxBS technology 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 Ultralow Methyl-Seq kit, the result is accurate, low-cost whole genome sequencing.

Features of the Ultralow Methyl-Seq with TrueMethyl oxBS:

  • DimerFree® library preparation
  • Integrated oxidative bisulfite conversion
  • Uniform whole genome coverage
  • Detection of 5mC and 5hmC with the same workflow
  • Directional libraries
  • All-in-one: From DNA to sequenceable library in a single kit

Applications

  • Whole genome bisulfite sequencing (WGBS)
  • Methyl-Seq
  • Oxidative bisulfite sequencing (oxBS-Seq)

 




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Tab 02 / Highlights
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Complete Methyl-Seq workflow in approximately 9 hours

  • Simple add and incubate workflow with minimal hands-on time
  • Barcoded bisulfite sequencing libraries from as little as 10 ng human DNA

Ovation Whole Blood Signal Correlation

Duplicate samples were prepared from 20 ng of human whole blood RNA using the Ovation Whole Blood Solution, according to the product user guides. RNA samples were isolated using the Agencourt RNAdvance™ Blood kit on the Beckman ArrayPlex. Targets were hybridized to Affymetrix GeneChip®U133_plus_2.0 arrays. Results of linear regression analysis show a strong signal correlation of R = 0.992, with 58% present calls, demonstrating very high reproducibility across transcript abundance with only 20 ng of input total whole blood RNA.

Obtain high quality results from very low inputs

  • DimerFree technology eliminates adaptor artifacts without the need for adaptor titration
  • Faster time to data with better reproducibility and consistency

 

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No adapter artifacts

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

Constant %MeCpG indicates lack of PCR bias

Mapping and %MeCpG are two metrics produced by Bismark. The % Mapping metric refers only to reads that map uniquely to the bisulfite converted genome. Reads that map to multiple locations are not included. Sequences that fail to map are less than 10% of all reads. %MeCpG remains relatively constant across the three libraries, indicating a lack of PCR bias, even as the number of PCR cycles increases from 12 cycles to 18 cycles.

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.

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
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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.

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

10 - 300 ng (100 - 300ng with TrueMethyl oxBS)




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Tab 04 / Faq
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FAQ

Input recommendations

Can I use FFPE or other degraded DNA as input into Tecan DNA-Seq library prep kits?

We recommend using high quality DNA with minimal degradation. However, moderately degraded DNA may also work with these kits. The A260:A280 ratio for DNA samples should be in excess of 1.8 and A260:A230 should be in excess of 2.0. Use of DNA samples with lower ratios may result in low library yield or compromised results.

General workflow

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

The bisulfite conversion reagents are included with the purchase of the Ultralow Methyl-Seq System with TrueMethyl oxBS (Part No. 0541-32 and 9513-A01) and the Ovation RRBS Methyl-Seq System 1-16 with TrueMethyl oxBS (Part No. 0553-32). Other commercially available bisulfite conversion kits may be suitable as well, but these have not been validated.

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.

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.

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

What is the expected yield of the amplified DNA library using the Ultralow Methyl-Seq?

The expected yield is at least 500 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.

Sequencing recommendations

What kind of sequencing primers can I use with your library?

The design of the Ultralow Methyl-Seq kit 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 Ultralow Methyl-Seq libraries be used with paired-end sequencing?

Yes, they can be used for both single- and paired-end sequencing. Special consideration should be given to the expected insert size in the paired-end assay. The expected distances between the 5’-most and 3’-most coordinates of paired-end reads will depend on the average fragment size of the insert pool.

Are the Ultralow Methyl-Seq kits compatible with all Illumina sequencing platforms?

A custom sequencing primer is required. Please follow the custom primer and low-diversity library recommendations for your specific sequencer.

What sequencers are compatible with your libraries?

Ultralow Methyl-Seq libraries are compatible with Illumina sequencing platforms. A custom read 1 primer is required. 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 Ultralow Methyl-Seq kit.




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Tab 05 / Literature
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Literature

Type
Title
No
User Guide
Ultralow Methyl-Seq with TrueMethyl oxBS Module
M01512 V2
Product Sheet
401213 V1
Safety Data Sheet
TrueMethyl oxBS Module
 
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