The sample types include but are not limited to gDNA, FFPE and blood samples. The DNA is then sheared into smaller fragments (~400 bp). The convenient one-step reaction setup involves incubating bisulfite converted template DNAwith a mixture of reaction buffer and REPLI-g DNA polymerase. Nat Biotechnol. d Global methylation levels of mESCs as measured by LC-MS and a panel of WGBS datasets. 2008;36:e150. M13mp18 (NEB) was used as a template. This enables effective DNA denaturation, which is necessary for complete cytosine conversion, and prevents the excessive DNA fragmentation usually associated with high temperature and low pH conditions of the bisulfite conversion reaction. 5d; Additional file2: Figure S8a). 2012;8:e1002750. PCR amplification was found to build on the over-represented methylated sequences and conversion artefacts, thus amplifying on the errors from BS treatment and becoming a major source of bias for both pre- and post-BS methods. No methylation-dependent coverage bias was detected with the Alkaline protocol, and an under-representation of CGIs was observed for the AT-biasing KAPA datasets. Promoters were defined as 1000 to +200 bp from the transcription start site, DMR coordinates were obtained from Seisenberger et al. a Coverage of dinucleotides in WGBS datasets from unmethylated and in vitro M.CviPI-methylated TKO DNA prepared with the Heat BS-seq protocol. Each dot represents a probe over 150 consecutive cytosines from the same genomic region in ESC and sperm. All datasets were deduplicated, consistent with common analysis pipelines, although this decreased the sequence and methylation bias, normally stronger in the raw duplicated data (unpublished observation). BS-treated fragments were eluted in the same final volume as the input and quantified for recovery three to four times for each sample. Ficz G, Branco MR, Seisenberger S, Santos F, Krueger F, Hore TA, et al. Dvoriantchikova G, Seemungal RJ, Ivanov DThe epigenetic plasticity of amphibian retinal pigment epithelium (RPE) allows them to regenerate the entire retina, a trait known to be absent in mammals. Ji L, Sasaki T, Sun X, Ma P, Lewis ZA, Schmitz RJ. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tanaka K, Okamoto A. Degradation of DNA by bisulfite treatment. The three protocols differ by denaturation method (Heat or Alkaline) or molarity of bisulfite (4.5 vs 9 M for Am-BS) but not by BS incubation temperature (6570 C). Only some of these considerations, however, remain relevant for NGS-based approaches (e.g. Investigation aiming at speeding-up the procedure for DNA methylation analysis. 2016. https://github.com/NellyOlova/BS_bias. 6d; Additional file2: Figure S10c). Our findings establish basic principles for understanding and minimising biases when designing and optimising WGBS strategies. We discuss mechanisms to avoid, predict or quantify biases and artefacts in future or for already available WGBS datasets. As NGS costs decrease, the WGBS approach becomes increasingly accessible for both fundamental and clinical research. 2016;11:e0152322. Averaged coverage values per method are expressed as log2 difference from the genomic expected and compared to a non-BS-treated control. Here we use Illumina HiSeq as our example to illustrate the workflow of WGBS. Incomplete conversion artefacts affect CG methylation and are a subject of over-amplification during PCR, but their most dramatic effect is on non-CG methylation. *p < 0.05, **p < 0.01, ***p<0.001, ****p<0.0001; error bars represent standard deviation. c Absolute quantification of unconverted cytosines in the unmethylated TKO mESC line, as measured by Heat and Alkaline BS-seq. The genome-wide analysis (scatter plots) was performed for two groups of datasets: 1) the panel of published mESC datasets from six WGBS methods generated by different studies or labs and used for the feature analyses (Additional file1); and 2) datasets generated by the same lab with PBAT and Heat BS-seq from four different biological samples (mESCs, sperm, blastocysts and oocytes) [29]. Cell. Kebschull JM, Zador AM. We find that the BS conversion step is the main trigger of biases, due to a selective and context-specific DNA degradation [24] and incomplete conversion efficiency, while subsequent PCR cycles primarily build on the effect of an already biased sequence composition. Download Safety Data Sheets for QIAGEN product components. Cell Rep. 2017;18:107989. 2009;6:2915. Typical DNA yields from the EpiTect Whole Bisulfitome Kit are 13 g per reaction, sufficient for up to 300 PCR analyses from just a single EpiTect bisulfite reaction. We then coupled the above BS conversion protocols to two strategies for the generation of WGBS libraries: 1) pre-BS, which adds sequencing adaptors by ligation before BS conversion [26, 27]; and 2) post-BS, which adds adaptors by random priming after BS conversion [28]. 1995;226(1):1616. 2013;14:R51. Recovery of BS-treated genomic DNA for the different BS conversion protocols and clean-up procedures was assessed in the same way as for the M13 fragments, but quantified with LC-MS. Article Exons and introns were defined with Ensembl-derived coordinates integrated in SeqMonk. PLoS One. We quantified the number of occurrences of each hexamer (or heptamer for A. thaliana) per read, as follows: TTAGGG (G-strand hexamer) or TTTTAA (BS converted version of the CCCTAA C-strand hexamer) for BS-seq and EpiGnome datasets; the reversed sequences CCCTAA(A) or (T)TTAAAA for PBAT and ampPBAT datasets (heptamers, in parentheses, were used for A. thaliana); TTAGGG, CCCTAA or TTTTAA for the non-BS converted control. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing. Notably, 5mC levels in amplification-free PBAT were not significantly different from those measured in genomic DNA (Fig. the moderately or variably methylated genomic regions (Fig. Genome Biology Some of the analyses, however, such as telomere and major satellite, were performed on raw (duplicated) data, since those sequences are not mappable. Although the commonly used Pfu Turbo Cx polymerase is not the worst performer among its counterparts [17], the KAPA HiFi family of polymerases used together with the PCR additive TMAC (tetramethylammonium chloride) have shown the best tolerance for AT-rich regions, albeit at the expense of higher error rates [18,19,20, 23]. Proc Natl Acad Sci U S A. The peaks represent the distribution of the reads. Cokus SJ, Feng S, Zhang X, Chen Z, Merriman B, Haudenschild CD, et al. Kumaki Y, Oda M, Okano M. QUMA: quantification tool for methylation analysis. 2. It has also been validated for bisulfite-converted library preparation from ChIP-derived samples in order to perform ChIP-Bis-Sequencing. 2014;10:112. ChIP Sequencing (Histone Modifications) qPCR-based Services. You are about to request a quote for our epigenomics services. Modules bam2nuc and filter_non_conversion are part of the Bismark suite [50], which is released under the GNU GPL v3 license. For the unmethylated DNA controls, library preparation was performed in the same way, with the following modifications: 1.0 g of DNA was sonicated and adaptor-ligated with Illumina TruSeq indexed adaptors, no BS conversion was performed, and amplification was done with the NEB Next 2 Phusion mix for six cycles, following the manufacturers instructions. Nucleic Acids Res. Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data. 2012;148:81631. 1b (Additional file2: Figure S3b), demonstrating a combined effect of the PCR and degradation biases (lowest for the KAPA datasets). In the EpiTect converted DNA, the four loci were represented similarly before and after amplification. This file contains comprehensive information about the product, including detailed operating instructions, reagent supplies and equipment to be used. More recently, BS treatment has been coupled with next generation sequencing (NGS) to yield reduced representation (RRBS) or whole genome (WGBS) data on the global genomic distribution of 5mC [4]. The tile distribution per G/C content is plotted in the background along the x-axis for reference. Comparison of whole-genome bisulfite sequencing library preparation Accessed 5 June 2017. Since its first use in 1992 [1], bisulfite (BS) sequencing of DNA has become the gold standard for analysis of DNA methylation. Google Scholar. 2013). DNA libraries are purified with magnetic beads and QCed with the Qubit quantification system and Agilent TapeStation. In this method, genomic DNA is treated with sodium bisulfite and then sequenced, providing single-base resolution of methylated cytosines in the genome. Comparison of whole-genome bisulfite sequencing library preparation The Premium Whole Genome Bisulfite . Hayatsu H, Negishi K, Shiraishi M. DNA methylation analysis: speedup of bisulfite-mediated deamination of cytosine in the genomic sequencing procedure. Proc Natl Acad Sci U S A. Arand J, Spieler D, Karius T, Branco MR, Meilinger D, Meissner A, et al. Genome-wide analysis of DNA methylation dynamics during early human development. e LC-MS measurement of total genomic 5mC levels in mouse ESC gDNA before (WT mESC) and after treatment with different BS-conversion protocols (Alkaline, Heat, Am-BS). 8 rxns. 2007;17:19125. With its newly introduced EpiTect products, QIAGEN makes available standardized, pre-analytical and analytical solutions, from DNA sample collection, stabilization and purification, to bisulfite conversion and real-time or end-point PCR methylation analysis or sequencing (see figure "Standardized workflows in epigenetics"). Farlik M, Sheffield NC, Nuzzo A, Datlinger P, Schnegger A, Klughammer J, et al. Identical aliquots were prepared from each fragment for BS treatment with the different protocols and an aliquot each was kept as an input control. For the telomere analyses we used only raw data reads, since the tandem hexamer units of the telomere are not mappable. Notably, we show that by minimizing amplification artefacts, the overall low bias of KAPA Uracil+ yields global methylation estimates close to those obtained with post-BS approaches (Fig. PDF Pico Methyl-Seq Library Prep Kit - Zymo Research Add Premium Whole Genome Bisulfite Sequencing (WGBS) kit to my shopping cart. 1998;264:12932. FastQC. In summary, all WGBS approaches show significant sequence coverage deviations in comparison to conventional WGS. not significant; error bars represent standard deviation. Google Scholar. [29], used for the whole genome analysis above. Positive y-axis values indicate the top strand and negative the bottom strand. PLoS Genet. Nature. Dotted black line represents the tile count distribution in G/C content. 2d), although localised and feature CG methylation values are nevertheless altered after amplification (Fig. The PCR was performed with Dream Taq DNA Polymerase (Thermo Scientific; 50 l volume, 200 nM primer, 200 M dNTPs, 1.25 units enzyme) with an initial step at 95 C for 2 min, 35 cycles of 30 s at 95 C, 20 s at 57 C, and 30 s (C, 5hmC) or 5 min (5mC) at 72 C, with a 7-min final step at 72 C (primers are listed in Additional file2: Table S2). Contribution of intragenic DNA methylation in mouse gametic DNA methylomes to establish oocyte-specific heritable marks. This analysis is integrated as the bam2nuc module in Bismark v0.16.0 [50]. 23andMe offers DNA testing with the most comprehensive ancestry breakdown, personalized health insights and more. The main method groups are additionally split into subgroups of Heat and Alkaline for KAPA and ampPBAT, as shown also in Additional file2: Figure S3a. These encompass biases associated with cloning and PCR, such as primer selectivity and design, polymerase sequence preferences and errors, and template switch (strand recombination) [3, 5, 6]. The ChIPd DNA was then further processed with the Premium WGBS Kit and sequenced. Significance is calculated by one-way ANOVA with Dunnetts multiple comparisons test on the absolute WGBS values (genomic + artefact) against the LC-MS value. 2010;5:479. The NEBNext Enzymatic Methyl-seq kit appeared to be the best option for whole-genome DNA methylation sequencing of high-quality DNA, closely followed by the Swift kit, which potentially works better for degraded samples. During bisulfite conversion, unmethylated cytosines are converted to uracil and later amplified as "T" in PCR, while 5-mC and 5-hmC are resistant to conversion and . The resulting DNA amplicons are sequenced with high sequencing coverage. Numbers indicate the mean value, error bars show the 1090 percentile. Krueger F, Andrews SR, Olova N. BS_bias scripts. The EpiTect Whole Bisulfitome Kit has outperformed amplification kits tested from other suppliers(see figures ", For whole genome amplification of bisulfite. Anal Biochem. a Presence of unconverted cytosines as percentage of total cytosine content, measured by LC-MS for three different BS-conversion protocols. Ficz G, Hore TA, Santos F, Lee HJ, Dean W, Arand J, et al. 5mC is represented as a percentage of all cytosines; the converted cytosines were measured as uracils in the BS converted samples. In order to exclude the non-telomere-derived TTTTAA and (T)TTAAAA hexa/heptamers from the BS treated data, therefore, all reads containing less than 5 units per read were removed from the analyses. To test the effect of cytosine modifications on DNA fragmentation, we generated 5mC- and 5hmC-modified C-poor and C-rich fragments. 2014;24:82130. Nature. To dissect polymerase differences, we have also included a pre-BS approach performed with the low-bias KAPA HiFi Uracil+ [23] to compare with the most commonly used Pfu Turbo Cx polymerase (Table2). Genereux DP, Johnson WC, Burden AF, Stger R, Laird CD. A more recent study suggested that the bias resulting from PCR in NGS stems primarily from factors such as stochasticity of amplification of low-copy sequences and polymerase errors [21]. We made probes over 150-cytosine-containing tiles over the whole genome for the mESC PBAT dataset (due to lowest coverage) and quantified CG methylation for these probes in PBAT sperm, as well as in the BS-seq datasets for both samples. Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies. These features of the KAPA profile were not affected by the BS conversion protocol used and the bias did not decrease in libraries generated with fewer PCR cycles (Additional file2: Figure S3a). Imprinted differentially methylated region, Liquid chromatography with mass-spectrometry, Reduced representation bisulfite sequencing. As targeted BS sequencing relies on primers that select for fully converted fragments [9], these results are more likely to reflect the real non-CG methylation distribution. The latter approach is particularly important for studies of model organisms with very low or questionable methylation levels near the detection limit, such as insects. Cytosines adjacent to methylated CpG sites can be partially resistant to conversion in genomic bisulfite sequencing leading to methylation artifacts. 5). 2022 BioMed Central Ltd unless otherwise stated. Because of its repetitive nature, the data for major satellite was not deduplciated following alignment. The whole genome amplification of the bisulfite-treated DNA was done using multiple displacement amplification (MDA). DNA degradation is strong in harsher BS conversion protocols that utilise high denaturation temperatures (Heat) or high BS molarity (Am-BS). During bisulfite conversion, unmethylated cytosines are converted to uracil and later amplified as T in PCR, while 5-mC and 5-hmC are resistant to conversion and are amplified as C. 2a, right panel). Add Premium WGBS Indexes - 6 to my shopping cart. Article 5c; Additional file2: Figure S7ac). Article Colonies were screened with Roches Taq DNA Polymerase (25 l volume, 300 nM primer, 200 M dNTPs, 1.25 units enzyme) at 94 C for 10 min, 35 cycles of 30 s at 94 C, 30 s at 55 C, and 30 s at 72 C, with a final 72 C for 10 min and sent for Sanger sequencing at Beckman Coulter Genomics. Pico Methyl-Seq Library Prep Kit Catalog Nos. WGBS is the gold standard for bisulfite based DNA methylation studies as the entire genome is sequenced with base-level detail into . Heat+KAPA and EpiGnome protocols are included only as WT values for reference, due to unavailability of corresponding unmethylated DNA datasets. 2011;27:15712. We then selected regions with 20% CG methylation difference between sperm and ESC in both the PBAT and BS-seq datasets and plotted the regions obtained with PBAT onto the BS-seq plot and vice versa. Chromatin Immunoprecipitation against the H3K27me3 mark was performed using the Diagenodes iDeal ChIP-seq Kit for Histones on a Gm12878 human cell line. Seqmonk. Genome-wide screening for DNA methylation - Library Preparation of bisulfite-converted DNA from whole genomes. 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