RRBS-Analyser: A Comprehensive Web Server for Reduced Representation Bisulfite Sequencing Data Analysis
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In reduced representation bisulfite sequencing (RRBS), genomic DNA is digested with the restriction enzyme and then subjected to next-generation sequencing, which enables detection and quantification of DNA methylation at whole-genome scale with low cost. However, the data processing, interpretation, and analysis of the huge amounts of data generated pose a bioinformatics challenge. We developed RRBS-Analyser, a comprehensive genome-scale DNA methylation analysis server based on RRBS data. RRBS-Analyser can assess sequencing quality, generate detailed statistical information, align the bisulfite-treated short reads to reference genome, identify and annotate the methylcytosines (5mCs) and associate them with different genomic features in CG, CHG, and CHH content. RRBS-Analyser supports detection, annotation, and visualization of differentially methylated regions (DMRs) for multiple samples from nine reference organisms. Moreover, RRBS-Analyser provides researchers with detailed annotation of DMR-containing genes, which will greatly aid subsequent studies. The input of RRBS-Analyser can be raw FASTQ reads, generic SAM format, or self-defined format containing individual 5mC sites. RRBS-Analyser can be widely used by researchers wanting to unravel the complexities of DNA methylome in the epigenetic community. RRBS-Analyser is freely available at http://122.228.158.106/RRBSAnalyser/.Keywords:
Analyser
Bisulfite sequencing
Bisulfite
1. RNAse-treated DNA (at least 1 μg, volume should not exceed 130 μl) 2. AMPure XP beads (Beckman Coulter, catalog number: A63880) 3. 200 proof Ethyl alcohol (Sigma-Aldrich, catalog number: 459844-500ML) 4. Illumina TruSeq kit (Illumina, catalog number: FC-121-2001) 5. Methylcode Invitrogen kit (Life Technologies, Invitrogen TM , catalog number: MECOV50) Note: Other bisulfite conversion kits can be used, but were not tested in this protocol. 6. T4 DNA ligase (NEB, catalog number: M0202T) 7. Pfu Cx Hotstart DNA polymerase (Agilent, catalog number: 600412) 8. TOPO Blunt cloning kit (Life Technologies, Invitrogen TM , catalog number: 450245) 9. TOPO TA cloning kit (Life Technologies, Invitrogen TM , catalog number: 450030) 10. Ex Taq DNA polymerase (TaKaRa, Clonetech, catalog number: RR001B) 11. 1 Kb-plus DNA ladder (Life Technologies, Invitrogen TM , catalog number: 10787-018) 12. Agarose 13. 1x TAE buffer (see Recipes)
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Bisulfite sequencing
Illumina Methylation Assay
Methylated DNA immunoprecipitation
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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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Methylated DNA immunoprecipitation
Sanger sequencing
CpG site
Sodium bisulfite
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Bisulfite
Bisulfite sequencing
Methylated DNA immunoprecipitation
Epigenome
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DNA methylation has emerged as an important regulator of development and disease, necessitating the design of more efficient and cost-effective methods for detecting and quantifying this epigenetic modification. Next-generation sequencing (NGS) techniques offer single base resolution of CpG methylation levels with high statistical significance, but are also high cost if performed genome-wide. Here, we describe a simplified targeted bisulfite sequencing approach in which DNA sequencing libraries are prepared following sodium bisulfite conversion and two rounds of PCR for target enrichment and sample barcoding, termed BisPCR(2).We have applied the BisPCR(2) technique to validate differential methylation at several type 2 diabetes risk loci identified in genome-wide studies of human islets. We confirmed some previous findings while not others, in addition to identifying novel differentially methylated CpGs at these genes of interest, due to the much higher depth of sequencing coverage in BisPCR(2) compared to prior array-based approaches.This study presents a robust, efficient, and cost-effective technique for targeted bisulfite NGS, and illustrates its utility by reanalysis of prior findings from genome-wide studies.
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So far, bisulfite sequencing has been considered as the "gold standard" method for the detection of DNA methylation. The results of whole-genome bisulfite next-generation sequencing (WG-BS-Seq) allowed scientists to conclude that the vast majority of CpG sites in genomic DNA from human and animal tissues are invariably hypermethylated. Only a small proportion (≤ 10-15%) of CpG sites was found to manifest variable degree of methylation between different tissues or among individuals. Moreover, such variably methylated CpG sites were believed to occur almost exclusively within extended clusters (differentially methylated regions, or DMRs).
5-Methylcytosine
Methylated DNA immunoprecipitation
Cytosine
Illumina Methylation Assay
DNA nanoball sequencing
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Bisulfite sequencing
Bisulfite
Illumina Methylation Assay
Methylated DNA immunoprecipitation
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Aim: Validation of sequencing-based DNA methylation data is an important step for meaningful translation of findings. However, there has been limited assessment of different platforms to validate methylation data from next generation sequencing. Methods: We performed a comparative methylation analysis between the genome-wide platform of reduced representation bisulfite sequencing with a targeted, Sequenom EpiTyper platform (four genes were analyzed in 15 cell lines covering 52 CpG sites). Results: We show that the accuracy of validation substantially improves if results from multiple and adjacent CpG sites are combined rather than at single CpG sites. We demonstrate increased read number improves accuracy of reduced representation bisulfite sequencing results. Further, by using series of replicates, we document variation in samples analyzed by Sequenom EpiTyper, indicating the importance of including replicates to increase precision. Conclusion: The results reveal potential sources of bias and provide a guideline for refining study design for DNA methylation analysis.
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Recent advances made in "omics" technologies are contributing to a revolution in livestock selection and breeding practices. Epigenetic mechanisms, including DNA methylation are important determinants for the control of gene expression in mammals. DNA methylation research will help our understanding of how environmental factors contribute to phenotypic variation of complex production and health traits. High-throughput sequencing is a vital tool for the comprehensive analysis of DNA methylation, and bisulfite-based strategies coupled with DNA sequencing allows for quantitative, site-specific methylation analysis at the genome level or genome wide. Reduced representation bisulfite sequencing (RRBS) and more recently whole genome bisulfite sequencing (WGBS) have proven to be effective techniques for studying DNA methylation in both humans and mice. Here we report the development of RRBS and WGBS for use in sheep, the first application of this technology in livestock species. Important technical issues associated with these methodologies including fragment size selection and sequence depth are examined and discussed.
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Illumina Methylation Assay
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Bisulfite sequencing
Bisulfite
Illumina Methylation Assay
Methylated DNA immunoprecipitation
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Bisulfite
Bisulfite sequencing
Epigenomics
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