BackgroundDNA methylation is an important epigenetic modification involved in many biological processes. Bisulfite treatment coupled with high-throughput sequencing provides an effective approach for studying genome-wide DNA methylation at base resolution. Libraries such as whole genome bisulfite sequencing (WGBS) and reduced represented bisulfite sequencing (RRBS) are widely used for generating DNA methylomes, demanding efficient and versatile tools for aligning bisulfite sequencing data.ResultsWe have developed BS-Seeker2, an updated version of BS Seeker, as a full pipeline for mapping bisulfite sequencing data and generating DNA methylomes. BS-Seeker2 improves mappability over existing aligners by using local alignment. It can also map reads from RRBS library by building special indexes with improved efficiency and accuracy. Moreover, BS-Seeker2 provides additional function for filtering out reads with incomplete bisulfite conversion, which is useful in minimizing the overestimation of DNA methylation levels. We also defined CGmap and ATCGmap file formats for full representations of DNA methylomes, as part of the outputs of BS-Seeker2 pipeline together with BAM and WIG files.ConclusionsOur evaluations on the performance show that BS-Seeker2 works efficiently and accurately for both WGBS data and RRBS data. BS-Seeker2 is freely available at http://pellegrini.mcdb.ucla.edu/BS_Seeker2/ and the Galaxy server.

Summary Global DNA demethylation in humans is a fundamental process that occurs in pre-implantation embryos and reversion to naïve ground state pluripotent stem cells (PSCs). However the extent of DNA methylation reprogramming in human germline cells is unknown. Here we performed whole-genome bisulfite sequencing (WGBS) and RNA-sequencing (RNA-Seq) of human prenatal germline cells from 53–137 days of development. We discovered that the transcriptome and methylome of human germline is distinct from both human PSCs and the inner cell mass (ICM) of human blastocysts. Using this resource to monitor the outcome of global DNA demethylation with reversion of primed PSCs to the naïve ground state, we uncovered hotspots of ultralow methylation at transposons that are protected from demethylation in the germline and ICM. Taken together the human germline serves as a valuable in vivo tool for monitoring the epigenome of cells that have emerged from a global DNA demethylation event.

Escherichia coli has been engineered to produce isobutanol, with titers reaching greater than the toxicity level. However, the specific effects of isobutanol on the cell have never been fully understood. Here, we aim to identify genotype–phenotype relationships in isobutanol response. An isobutanol-tolerant mutant was isolated with serial transfers. Using whole-genome sequencing followed by gene repair and knockout, we identified five mutations (acrA, gatY, tnaA, yhbJ, and marCRAB) that were primarily responsible for the increased isobutanol tolerance. We successfully reconstructed the tolerance phenotype by combining deletions of these five loci, and identified glucosamine-6-phosphate as an important metabolite for isobutanol tolerance, which presumably enhanced membrane synthesis. The isobutanol-tolerant mutants also show increased tolerance to n-butanol and 2-methyl-1-butanol, but showed no improvement in ethanol tolerance and higher sensitivity to hexane and chloramphenicol than the parental strain. These results suggest that C4, C5 alcohol stress impacts the cell differently compared with the general solvent or antibiotic stresses. Interestingly, improved isobutanol tolerance did not increase the final titer of isobutanol production.

DNA methylation is an epigenetic modification that plays an important role in regulating gene expression and therefore a broad range of biological processes and diseases. DNA methylation is tissue-specific, dynamic, sequence-context-dependent and trans-generationally heritable, and these complex patterns of methylation highlight the significance of profiling DNA methylation to answer biological questions. In this review, we surveyed major methylation assays, along with comparisons and biological examples, to provide an overview of DNA methylation profiling techniques. The advances in microarray and sequencing technologies make genome-wide profiling possible at a single-nucleotide or even a single-cell resolution. These profiling approaches vary in many aspects, such as DNA input, resolution, genomic region coverage, and bioinformatics analysis, and selecting a feasible method requires knowledge of these methods. We first introduce the biological background of DNA methylation and its pattern in plants, animals and fungi. We present an overview of major experimental approaches to profiling genome-wide DNA methylation and hydroxymethylation and then extend to the single-cell methylome. To evaluate these methods, we outline their strengths and weaknesses and perform comparisons across the different platforms. Due to the increasing need to compute high-throughput epigenomic data, we interrogate the computational pipeline for bisulfite sequencing data and also discuss the concept of identifying differentially methylated regions (DMRs). This review summarizes the experimental and computational concepts for profiling genome-wide DNA methylation, followed by biological examples. Overall, this review provides researchers useful guidance for the selection of a profiling method suited to specific research questions.

SignificanceWe describe the spatial and temporal profiles of soybean and Arabidopsis seed methylomes during development. CHH methylation increases globally from fertilization through dormancy in all seed parts, decreases following germination, and targets primarily transposons. By contrast, CG- and CHG-context methylation remains constant throughout seed development. Mutant seeds lacking non-CG methylation develop normally, but have a set of up-regulated transposon RNAs suggesting that the CHH methylation increase may be a failsafe mechanism to reinforce transposon silencing. Major classes of seed genes have similar methylation profiles, whether they are active or not. Our results suggest that soybean and Arabidopsis seed methylomes are similar, and that DNA methylation does not play a significant role in regulating many genes important for seed development.