Single Base-Resolution Methylome of the Dizygotic Sheep

Sheep is an important livestock in the world for meat, dairy and wool production. The third version of sheep reference genome has been recently assembled, but sheep DNA methylome has not been profiled yet. In this study, we report the comprehensive sheep methylome with 94.38% cytosine coverage at single base resolution by sequencing DNA samples from Longissimus dorsi of dizygotic Sunit sheep, which were bred in different habitats. We also compared methylomes between the twin sheep. DNA methylation status at genome-scale differentially methylated regions (DMRs), functional genomic regions and 248 DMR-containing genes were identified between the twin sheep. Gene ontology (GO) and KEGG annotations of these genes were performed to discover computationally predicted function. Lipid metabolism, sexual maturity and tumor-associated categories were observed to significantly enrich DMR-containing genes. These findings could be used to illustrate the relationship between phenotypic variations and gene methylation patterns.     

Systematic assessment of reduced representation bisulfite sequencing to human blood samples: A promising method for large-sample-scale epigenomic studies

Complementary to the time- and cost-intensive direct bisulfite sequencing, we applied reduced representation bisulfite sequencing (RRBS) to the human peripheral blood mononuclear cells (PBMC) from YH, the Asian individual whose genome and epigenome has been deciphered in the YH project and systematically assessed the genomic coverage, coverage depth and reproducibility of this technology as well as the concordance of DNA methylation levels measured by RRBS and direct bisulfite sequencing for the detected CpG sites. Our result suggests that RRBS can cover more than half of CpG islands and promoter regions with a good coverage depth and the proportion of the CpG sites covered by the biological replicates reaches 80-90%, indicating good reproducibility. Given a smaller data quantity, RRBS enjoys much better coverage depth than direct bisulfite sequencing and the concordance of DNA methylation levels between the two methods is high. It can be concluded that RRBS is a time and cost-effective sequencing method for unbiased DNA methylation profiling of CpG islands and promoter regions in a genome-wide scale and it is the method of choice to assay certain genomic regions for multiple samples in a rapid way.     

Improved reduced representation bisulfite sequencing for epigenomic profiling of clinical samples

Background

DNA methylation plays crucial roles in epigenetic gene regulation in normal development and disease pathogenesis. Efficient and accurate quantification of DNA methylation at single base resolution can greatly advance the knowledge of disease mechanisms and be used to identify potential biomarkers. We developed an improved pipeline based on reduced representation bisulfite sequencing (RRBS) for cost-effective genome-wide quantification of DNA methylation at single base resolution. A selection of two restriction enzymes (Taq^αI and MspI) enables a more unbiased coverage of genomic regions of different CpG densities. We further developed a highly automated software package to analyze bisulfite sequencing results from the Solexa GAIIx system.

Results

With two sequencing lanes, we were able to quantify ~1.8 million individual CpG sites at a minimum sequencing depth of 10. Overall, about 76.7% of CpG islands, 54.9% of CpG island shores and 52.2% of core promoters in the human genome were covered with at least 3 CpG sites per region.

Conclusions

With this new pipeline, it is now possible to perform whole-genome DNA methylation analysis at single base resolution for a large number of samples for understanding how DNA methylation and its changes are involved in development, differentiation, and disease pathogenesis.     

Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling

Genome-wide mapping of 5-methylcytosine is of broad interest to many fields of biology and medicine. A variety of methods have been developed, and several have recently been advanced to genome-wide scale using arrays and next-generation sequencing approaches. We have previously reported reduced representation bisulfite sequencing (RRBS), a bisulfite-based protocol that enriches CG-rich parts of the genome, thereby reducing the amount of sequencing required while capturing the majority of promoters and other relevant genomic regions. The approach provides single-nucleotide resolution, is highly sensitive and provides quantitative DNA methylation measurements. This protocol should enable any standard molecular biology laboratory to generate RRBS libraries of high quality. Briefly, purified genomic DNA is digested by the methylation-insensitive restriction enzyme MspI to generate short fragments that contain CpG dinucleotides at the ends. After end-repair, A-tailing and ligation to methylated Illumina adapters, the CpG-rich DNA fragments (40-220 bp) are size selected, subjected to bisulfite conversion, PCR amplified and end sequenced on an Illumina Genome Analyzer. Note that alignment and analysis of RRBS sequencing reads are not covered in this protocol. The extremely low input requirements (10-300 ng), the applicability of the protocol to formalin-fixed and paraffin-embedded samples, and the technique's single-nucleotide resolution extends RRBS to a wide range of biological and clinical samples and research applications. The entire process of RRBS library construction takes ～9 d.     

DMRFusion: A differentially methylated region detection tool based on the ranked fusion method

DNA methylation is an important epigenetic modification involved in many biological processes and diseases. Computational analysis of differentially methylated regions (DMRs) could explore the underlying reasons of methylation. DMRFusion is presented as a useful tool for comprehensive DNA methylation analysis of DMRs on methylation sequencing data. This tool is designed base on the integration of several ranking methods; Information gain, Between versus within Class scatter ratio, Fisher ratio, Z-score and Welch's t-test. In this study, DMRFusion on reduced representation bisulfite sequencing (RRBS) data in chronic lymphocytic leukemia cancer displayed 30 nominated regions and CpG sites with a maximum methylation difference detected in the hypermethylation DMRs. We realized that DMRFusion is able to process methylation sequencing data in an efficient and accurate manner and to provide annotation and visualization for DMRs with high fold difference score (p-value and FDR < 0.05 and type I error: 0.04).     

Mapping the zebrafish brain methylome using reduced representation bisulfite sequencing

Reduced representation bisulfite sequencing (RRBS) has been used to profile DNA methylation patterns in mammalian genomes such as human, mouse and rat. The methylome of the zebrafish, an important animal model, has not yet been characterized at base-pair resolution using RRBS. Therefore, we evaluated the technique of RRBS in this model organism by generating four single-nucleotide resolution DNA methylomes of adult zebrafish brain. We performed several simulations to show the distribution of fragments and enrichment of CpGs in different in silico reduced representation genomes of zebrafish. Four RRBS brain libraries generated 98 million sequenced reads and had higher frequencies of multiple mapping than equivalent human RRBS libraries. The zebrafish methylome indicates there is higher global DNA methylation in the zebrafish genome compared with its equivalent human methylome. This observation was confirmed by RRBS of zebrafish liver. High coverage CpG dinucleotides are enriched in CpG island shores more than in the CpG island core. We found that 45% of the mapped CpGs reside in gene bodies, and 7% in gene promoters. This analysis provides a roadmap for generating reproducible base-pair level methylomes for zebrafish using RRBS and our results provide the first evidence that RRBS is a suitable technique for global methylation analysis in zebrafish.     

癌症相关的DNA甲基化连锁区域

DNA甲基化是重要的表观遗传标记之一,在转录调控中起直接作用。DNA甲基化的异常与癌症的发生发展密切相关。高通量测序使得在单碱基分辨率下检测全基因组的DNA甲基化水平成为可能。本文基于临近CpGs位点甲基化水平的相关性挖掘DNA甲基化连锁区域。结果发现DNA甲基化连锁区域的甲基化水平和模式在癌症中存在异常,而且显著富集到分化/发育相关的生物学功能。DNA甲基化连锁区域的挖掘有助于对具有生物学功能的表观遗传标记的进一步理解,有助于对癌症诊断的表观遗传标记的挖掘。     

Genome-Wide Analysis of DNA Methylation Dynamics during Early Human Development

DNA methylation is globally reprogrammed during mammalian preimplantation development, which is critical for normal development. Recent reduced representation bisulfite sequencing (RRBS) studies suggest that the methylome dynamics are essentially conserved between human and mouse early embryos. RRBS is known to cover 5–10% of all genomic CpGs, favoring those contained within CpG-rich regions. To obtain an unbiased and more complete representation of the methylome during early human development, we performed whole genome bisulfite sequencing of human gametes and blastocysts that covered>70% of all genomic CpGs. We found that the maternal genome was demethylated to a much lesser extent in human blastocysts than in mouse blastocysts, which could contribute to an increased number of imprinted differentially methylated regions in the human genome. Global demethylation of the paternal genome was confirmed, but SINE-VNTR-Alu elements and some other tandem repeat-containing regions were found to be specifically protected from this global demethylation. Furthermore, centromeric satellite repeats were hypermethylated in human oocytes but not in mouse oocytes, which might be explained by differential expression of de novo DNA methyltransferases. These data highlight both conserved and species-specific regulation of DNA methylation during early mammalian development. Our work provides further information critical for understanding the epigenetic processes underlying differentiation and pluripotency during early human development.     

Epigenetic effects of parasites and pesticides on captive and wild nestling birds

Anthropogenic changes to the environment challenge animal populations to adapt to new conditions and unique threats. While the study of adaptation has focused on genetic variation, epigenetic mechanisms may also be important. DNA methylation is sensitive to environmental stressors, such as parasites and pesticides, which may affect gene expression and phenotype. We studied the effects of an invasive ectoparasite, Philornis downsi, on DNA methylation of Galápagos mockingbirds (Mimus parvulus). We used the insecticide permethrin to manipulate P. downsi presence in nests of free‐living mockingbirds and tested for effects of parasitism on nestling mockingbirds using epiGBS, a reduced‐representation bisulfite sequencing (RRBS) approach. To distinguish the confounding effects of insecticide exposure, we conducted a matching experiment exposing captive nestling zebra finches (Taeniopygia guttata) to permethrin. We used zebra finches because they were the closest model organism to mockingbirds that we could breed in controlled conditions. We identified a limited number of differentially methylated cytosines (DMCs) in parasitized versus nonparasitized mockingbirds, but the number was not more than expected by chance. In contrast, we saw clear effects of permethrin on methylation in captive zebra finches. DMCs in zebra finches paralleled documented effects of permethrin exposure on vertebrate cellular signaling and endocrine function. Our results from captive birds indicate a role for epigenetic processes in mediating sublethal nontarget effects of pyrethroid exposure in vertebrates. Environmental conditions in the field were more variable than the laboratory, which may have made effects of both parasitism and permethrin harder to detect in mockingbirds. RRBS approaches such as epiGBS may be a cost‐effective way to characterize genome‐wide methylation profiles. However, our results indicate that ecological epigenetic studies in natural populations should consider the number of cytosines interrogated and the depth of sequencing in order to have adequate power to detect small and variable effects.     

Allele-specific DNA methylation analyses associated with siRNAs in Arabidopsis hybrids

Accumulating evidence has suggested that epigenetic marks including DNA methylation,small RNA and histone modification may involve hybrid vigor in plants.However,knowledge about how epigenetic marks in hybrids regulate gene expression is still limited.Based on genome-wide DNA methylation landscapes of Arabidopsis thaliana Ler and C24 ecotypes and their reciprocal F1 hybrids which were obtained in our previous work,we analyzed allele-specific DNA methylation and distinguished cis-and trans-regulated DNA methylation in hybrids.Our study indicated that both cis-and trans-regulated DNA methylation played roles in hybrids,when cis-regulation played a major role in CG methylation and trans-regulation played major roles in CHG and CHH methylation.In addition,we observed correlations between trans-regulated DNA methylation and siRNA densities.Enriched siRNA regions were significantly concurrent with highly trans-regulated DNA methylation regions.Our results illustrated DNA methylation regulation patterns integrated with siRNAs in Arabidopsis hybrids,and shed light on understanding the mechanism of epigenetic reprogramming for hybrid vigor.     

Reduced representation bisulfite sequencing design for assessing the methylation of human CpG islands in large samples

The reduced representation bisulfite sequencing (RRBS) method has been developed for the high-throughput analysis of DNA methylation based on the sequencing of genomic libraries treated with sodium bisulfite by next-generation approaches. In contrast to whole-genome sequencing, the RRBS approach elaborates specific endonucleases to prepare libraries in order to produce pools of CpG-rich DNA fragments. The original RRBS technology based on the use of the MspI libraries allows one to increase the relative number of CpG islands in the pools of genomic fragments compared to whole-genome bisulfite sequencing. Nevertheless, this technology is rarely used due to the high cost compared with bisulfite methylation analysis with hybridization microarrays and significant residual amount of data represented by the sequences of genomic repeats that complicates the alignment and is not of particular interest for developing DNA methylation markers, which is often the main goal of biomedical research. We have developed an algorithm for estimating the likelihood that recognition sites of restriction endonucleases will be represented in CpG islands and present a method of reducing the effective size of the RRBS library without a significant loss of the CpG islands based on the use of the XmaI endonuclease for library preparation. In silico analysis demonstrates that the optimum range of the XmaI-RRBS fragment lengths is 110–200 base pairs. The sequencing of this library allows one to assess the methylation status of over 125000 CpG dinucleotides, of which over 90000 belong to CpG islands.

     

Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis

We describe a large-scale random approach termed reduced representation bisulfite sequencing (RRBS) for analyzing and comparing genomic methylation patterns. BglII restriction fragments were size-selected to 500–600 bp, equipped with adapters, treated with bisulfite, PCR amplified, cloned and sequenced. We constructed RRBS libraries from murine ES cells and from ES cells lacking DNA methyltransferases Dnmt3a and 3b and with knocked-down (kd) levels of Dnmt1 (Dnmt[1^kd,3a^−/−,3b^−/−]). Sequencing of 960 RRBS clones from Dnmt[1^kd,3a^−/−,3b^−/−] cells generated 343 kb of non-redundant bisulfite sequence covering 66212 cytosines in the genome. All but 38 cytosines had been converted to uracil indicating a conversion rate of >99.9%. Of the remaining cytosines 35 were found in CpG and 3 in CpT dinucleotides. Non-CpG methylation was >250-fold reduced compared with wild-type ES cells, consistent with a role for Dnmt3a and/or Dnmt3b in CpA and CpT methylation. Closer inspection revealed neither a consensus sequence around the methylated sites nor evidence for clustering of residual methylation in the genome. Our findings indicate random loss rather than specific maintenance of methylation in Dnmt[1^kd,3a^−/−,3b^−/−] cells. Near-complete bisulfite conversion and largely unbiased representation of RRBS libraries suggest that random shotgun bisulfite sequencing can be scaled to a genome-wide approach.     

Performances of Different Fragment Sizes for Reduced Representation Bisulfite Sequencing in Pigs

Background

Reduced representation bisulfite sequencing (RRBS) has been widely used to profile genome-scale DNA methylation in mammalian genomes. However, the applications and technical performances of RRBS with different fragment sizes have not been systematically reported in pigs, which serve as one of the important biomedical models for humans. The aims of this study were to evaluate capacities of RRBS libraries with different fragment sizes to characterize the porcine genome.

Results

We found that the MspI-digested segments between 40 and 220 bp harbored a high distribution peak at 74 bp, which were highly overlapped with the repetitive elements and might reduce the unique mapping alignment. The RRBS library of 110–220 bp fragment size had the highest unique mapping alignment and the lowest multiple alignment. The cost-effectiveness of the 40–110 bp, 110–220 bp and 40–220 bp fragment sizes might decrease when the dataset size was more than 70, 50 and 110 million reads for these three fragment sizes, respectively. Given a 50-million dataset size, the average sequencing depth of the detected CpG sites in the 110–220 bp fragment size appeared to be deeper than in the 40–110 bp and 40–220 bp fragment sizes, and these detected CpG sties differently located in gene- and CpG island-related regions.

Conclusions

In this study, our results demonstrated that selections of fragment sizes could affect the numbers and sequencing depth of detected CpG sites as well as the cost-efficiency. No single solution of RRBS is optimal in all circumstances for investigating genome-scale DNA methylation. This work provides the useful knowledge on designing and executing RRBS for investigating the genome-wide DNA methylation in tissues from pigs.