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Fast and scalable solution for producing Methyl-seq libraries for Whole genome methylation profiling.
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.
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 within 6 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.
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.
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.
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.
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.
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
10 - 300 ng (100 - 300ng with TrueMethyl oxBS)
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.
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.
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.
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.
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.
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:
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.
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.
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.
A custom sequencing primer is required. Please follow the custom primer and low-diversity library recommendations for your specific sequencer.
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.
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 Ultralow Methyl-Seq kit.
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For research use only. Not for use in diagnostic procedures.