Decision: MethylScore, a pipeline for accurate and context-aware identification of differentially methylated regions from population-scale plant whole-genome bisulfite sequencing data — R2/PR13
Patrick HütherJörg HagmannAdam NunnIoanna KakoulidouRahul PisupatiDavid LangenbergerDetlef WeigelFrank JohannesSebastian J. SchultheißClaude Becker
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Abstract:
Whole-genome bisulfite sequencing (WGBS) is the standard method for profiling DNA methylation at single-nucleotide resolution. Different tools have been developed to extract differentially methylated regions (DMRs), often built upon assumptions from mammalian data. Here, we present MethylScore, a pipeline to analyse WGBS data and to account for the substantially more complex and variable nature of plant DNA methylation. MethylScore uses an unsupervised machine learning approach to segment the genome by classification into states of high and low methylation. It processes data from genomic alignments to DMR output and is designed to be usable by novice and expert users alike. We show how MethylScore can identify DMRs from hundreds of samples and how its data-driven approach can stratify associated samples without prior information. We identify DMRs in the A. thaliana 1,001 Genomes dataset to unveil known and unknown genotype–epigenotype associations .Keywords:
Differentially methylated regions
Bisulfite sequencing
Bisulfite
Abstract DNA methylation is measured using bisulfite sequencing (BS-seq). Bisulfite conversion can have low efficiency and a DNA sample is then processed multiple times generating DNA libraries with different bisulfite conversion rates. Libraries with low conversion rates are excluded from analysis resulting in reduced coverage and increased costs. We present a method and software, LuxRep, that accounts for technical replicates from different bisulfite-converted DNA libraries. We show that including replicates with low bisulfite conversion rates generates more accurate estimates of methylation levels and differentially methylated sites. Availability An implementation of the method is available at https://github.com/tare/LuxGLM/tree/master/LuxRep Contact maia.malonzo@aalto.fi
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Introduction Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity. Methods Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood. Results The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood. Conclusion Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed. Finally, we showed that bisulfite amplicon sequencing is a suitable approach for methylation analysis of targeted regions.
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Bisulfite
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Methylated DNA immunoprecipitation
Illumina Methylation Assay
genomic DNA
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Abstract Heat stress affects muscle development and meat quality in food animals; however, little is known regarding its regulatory mechanisms at the epigenetic level, such as via DNA methylation. In this study, we aimed to compare the DNA methylation profiles between control and heat-stressed pigs to identify candidate genes for skeletal muscle development and meat quality. Whole-genome bisulfite sequencing was used to investigate the genome-wide DNA methylation patterns in the longissimus dorsi muscles of the pigs. Both groups showed similar proportions of methylation at CpG sites but exhibited different proportions at non-CpG sites. A total of 57,147 differentially methylated regions were identified between the two groups, which corresponded to 1,422 differentially methylated genes. Gene ontogeny and KEGG pathway analyses indicated that these were mainly involved in energy and lipid metabolism, cellular defense and stress responses and calcium signaling pathways. This study revealed the global DNA methylation pattern of pig muscle between normal and heat stress conditions. The result of this study might contribute to a better understanding of epigenetic regulation in pig muscle development and meat quality.
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Illumina Methylation Assay
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Aim: Our purpose is to improve the conventional procedures for bisulfite conversion used to detect 5-methylcytosine in DNA. Methods: Impacts of different bisulfite salts, bisulfite conversion temperature, antioxidants and denaturants on DNA conversion and degradation were assessed by methylation-sensitive melt curve analysis. The modified method was tested on different genes and the conversion efficiency was analyzed by bisulfite sequencing. Results: We developed a modified bisulfite conversion method that completes this process within 2 h. We demonstrate that high temperature denaturation is the major cause for DNA degradation, and the addition of ethylene glycol dimethyl ether is an effective way to accelerate the bisulfite conversion. The conversion efficiency is comparable to many other commercial kits. Conclusion: Our modified bisulfite conversion method is simple, cost efficient and less time consuming and is compatible with different genes and samples, thus has a great potential for the future research and clinical applications.
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Bisulfite is known to deaminate cytosine in nucleic acids, while 5-methylcytosine resists this bisulfite action. For this reason, bisulfite treatment has been used for detecting 5-methylcytosine in DNA, a minor component of eukaryotic DNA, presently recognized as playing an important role in the control of gene function. This procedure, called bisulfite genomic sequencing, is a principal method for the analysis of DNA methylation in various biological phenomena, including human diseases such as cancer. This unit describes an efficient procedure utilizing a newly developed high-concentration bisulfite solution. Protocols for this methodology are supplemented with discussions focused on chemical aspects of the bisulfite treatment.
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DNA methylation analyses usually require a preceding bisulfite conversion of the DNA. The choice of an appropriate kit for a specific application should be based on the specific performance requirements with regard to the respective sample material. In this study, the performance of nine kits was evaluated: EpiTect Fast FFPE Bisulfite Kit, EpiTect Bisulfite Kit, EpiTect Fast DNA Bisulfite Kit (Qiagen), EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Direct Kit, EZ DNA Methylation-Lightning Kit (Zymo Research), innuCONVERT Bisulfite All-In-One Kit, innuCONVERT Bisulfite Basic Kit, innuCONVERT Bisulfite Body Fluids Kit (Analytik Jena). The kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis. All kits yielded highly pure DNA suitable for PCR analyses without PCR inhibition. Significantly higher yields were obtained when using the EZ DNA Methylation-Gold Kit and the innuCONVERT Bisulfite kits. Conversion efficiency ranged from 98.7% (EpiTect Bisulfite Kit) to 99.9% (EZ DNA Methylation-Direct Kit). The inappropriate conversion of methylated cytosines to thymines varied between 0.9% (innuCONVERT Bisulfite kits) and 2.7% (EZ DNA Methylation-Direct Kit). Time-to-result ranged from 131 min (innuCONVERT kits) to 402 min (EpiTect Bisulfite Kit). Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits). Highest yields from formalin-fixed and paraffin-embedded (FFPE) tissue sections without prior extraction were obtained using the innuCONVERT Bisulfite All-In-One Kit while the EZ DNA Methylation-Direct Kit yielded DNA with only low PCR-amplifiability. The innuCONVERT Bisulfite All-In-One Kit exhibited the highest versatility regarding different input sample materials (extracted DNA, tissue, FFPE tissue, cell lines, urine sediment, and cellular fractions of bronchial aspirates, pleural effusions, ascites). The innuCONVERT Bisulfite Body Fluids Kit allowed for the analysis of 3 ml plasma, serum, ascites, pleural effusions and urine.
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