BiQ Analyzer HT: locus-specific analysis of DNA methylation by high-throughput bisulfite sequencing

Bisulfite sequencing is a widely used method for measuring DNA methylation in eukaryotic genomes. The assay provides single-base pair resolution and, given sufficient sequencing depth, its quantitative accuracy is excellent. High-throughput sequencing of bisulfite-converted DNA can be applied either genome wide or targeted to a defined set of genomic loci (e.g. using locus-specific PCR primers or DNA capture probes). Here, we describe BiQ Analyzer HT (http://biq-analyzer-ht.bioinf.mpi-inf.mpg.de/), a user-friendly software tool that supports locus-specific analysis and visualization of high-throughput bisulfite sequencing data. The software facilitates the shift from time-consuming clonal bisulfite sequencing to the more quantitative and cost-efficient use of high-throughput sequencing for studying locus-specific DNA methylation patterns. In addition, it is useful for locus-specific visualization of genome-wide bisulfite sequencing data

Genome-wide quantitative analysis of DNA methylation from bisulfite sequencing data

Summary: Here we present the open-source R/Bioconductor software package BEAT (BS-Seq Epimutation Analysis Toolkit). It implements all bioinformatics steps required for the quantitative high-resolution analysis of DNA methylation patterns from bisulfite sequencing data, including the detection of regional epimutation events, i.e. loss or gain of DNA methylation at CG positions relative to a reference. Using a binomial mixture model, the BEAT package aggregates methylation counts per genomic position, thereby compensating for low coverage, incomplete conversion and sequencing errors. Availability and implementation: BEAT is freely available as part of Bioconductor at www.bioconductor.org/packages/devel/bioc/html/BEAT.html. The package is distributed under the GNU Lesser General Public License 3.0. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online

Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications.

Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA binding domain sequencing (MBD-seq). We applied all four methods to biological replicates of human embryonic stem cells to assess their genome-wide CpG coverage, resolution, cost, concordance and the influence of CpG density and genomic context. The methylation levels assessed by the two bisulfite methods were concordant (their difference did not exceed a given threshold) for 82% for CpGs and 99% of the non-CpG cytosines. Using binary methylation calls, the two enrichment methods were 99% concordant and regions assessed by all four methods were 97% concordant. We combined MeDIP-seq with methylation-sensitive restriction enzyme (MRE-seq) sequencing for comprehensive methylome coverage at lower cost. This, along with RNA-seq and ChIP-seq of the ES cells enabled us to detect regions with allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression

{BiQ} Analyzer {HiMod}: An Interactive Software Tool for High-throughput Locus-specific Analysis of 5-Methylcytosine and its Oxidized Derivatives

Recent data suggest important biological roles for oxidative modifications of methylated cytosines, specifically hydroxymethylation, formylation and carboxylation. Several assays are now available for profiling these DNA modifications genome-wide as well as in targeted, locus-specific settings. Here we present BiQ Analyzer HiMod, a user-friendly software tool for sequence alignment, quality control and initial analysis of locus-specific DNA modification data. The software supports four different assay types, and it leads the user from raw sequence reads to DNA modification statistics and publication-quality plots. BiQ Analyzer HiMod combines well-established graphical user interface of its predecessor tool, BiQ Analyzer HT, with new and extended analysis modes. BiQ Analyzer HiMod also includes updates of the analysis workspace, an intuitive interface, a custom vector graphics engine and support of additional input and output data formats. The tool is freely available as a stand-alone installation package from http://biq-analyzer-himod.bioinf.mpi-inf.mpg.de/

Comprehensive profiling of zebrafish hepatic proximal promoter CpG island methylation and its modification during chemical carcinogenesis

Background\ud DNA methylation is an epigenetic mechanism associated with regulation of gene expression and it is modulated during chemical carcinogenesis. The zebrafish is increasingly employed as a human disease model; however there is a lack of information on DNA methylation in zebrafish and during fish tumorigenesis. \ud \ud Results\ud A novel CpG island tiling array containing 44,000 probes, in combination with immunoprecipitation of methylated DNA, was used to achieve the first comprehensive methylation profiling of normal adult zebrafish liver. DNA methylation alterations were detected in zebrafish liver tumors induced by the environmental carcinogen 7, 12-dimethylbenz(a)anthracene. Genes significantly hypomethylated in tumors were associated particularly with proliferation, glycolysis, transcription, cell cycle, apoptosis, growth and metastasis. Hypermethylated genes included those associated with anti-angiogenesis and cellular adhesion. Of 49 genes that were altered in expression within tumors, and which also had appropriate CpG islands and were co-represented on the tiling array, approximately 45% showed significant changes in both gene expression and methylation. \ud \ud Conclusion\ud The functional pathways containing differentially methylated genes in zebrafish hepatocellular carcinoma have also been reported to be aberrantly methylated during tumorigenesis in humans. These findings increase the confidence in the use of zebrafish as a model for human cancer in addition to providing the first comprehensive mapping of DNA methylation in the normal adult zebrafish liver. \ud \u

Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions

Background: Cytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome- wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material. Methods: We established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species. Results: As a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant-RRBS reproducibly covers commonly up to one fourth of the cytosine positions in the rice genome when using MspI-DpnII within a group of five biological replicates of a line. The method predominantly detects cytosine methylation in putative promoter regions and not-annotated regions in rice. Conclusions: Plant-RRBS offers high-throughput and broad, genome- dispersed methylation detection by effective read number generation obtained from reproducibly covered genome fractions using optimized endonuclease combinations, facilitating comparative analyses of multi-sample studies for cytosine methylation and transgenerational stability in experimental material and plant breeding populations

Methylation Status of Imprinted Genes and Repetitive Elements in Sperm DNA from Infertile Males

Stochastic, environmentally and/or genetically induced disturbances in the genome-wide epigenetic reprogramming processes during male germ-cell development may contribute to male infertility. To test this hypothesis, we have studied the methylation levels of 2 paternally (H19 and GTL2) and 5 maternally methylated (LIT1, MEST, NESPAS, PEG3, and SNRPN) imprinted genes, as well as of ALU and LINE1 repetitive elements in 141 sperm samples, which were used for assisted reproductive technologies (ART), including 106 couples with strictly male-factor or combined male and female infertility and 28 couples with strictly female-factor infertility. Aberrant methylation imprints showed a significant association with abnormal semen parameters, but did not seem to influence ART outcome. Repeat methylation also differed significantly between sperm samples from infertile and presumably fertile males. However, in contrast to imprinted genes, ALU methylation had a significant impact on pregnancy and live-birth rate in couples with male-factor or combined infertility. ALU methylation was significantly high-er in sperm samples leading to pregnancy and live-birth than in those that did not. Sperm samples leading to abortions showed significantly lower ALU methylation levels than those leading to the birth of a baby. Copyright (C) 2011 S. Karger AG, Base

Epigenetic aging signatures in mice livers are slowed by dwarfism, calorie restriction and rapamycin treatment

Background: Global but predictable changes impact the DNA methylome as we age, acting as a type of molecular clock. This clock can be hastened by conditions that decrease lifespan, raising the question of whether it can also be slowed, for example, by conditions that increase lifespan. Mice are particularly appealing organisms for studies of mammalian aging; however, epigenetic clocks have thus far been formulated only in humans. Results: We first examined whether mice and humans experience similar patterns of change in the methylome with age. We found moderate conservation of CpG sites for which methylation is altered with age, with both species showing an increase in methylome disorder during aging. Based on this analysis, we formulated an epigenetic-aging model in mice using the liver methylomes of 107 mice from 0.2 to 26.0 months old. To examine whether epigenetic aging signatures are slowed by longevity-promoting interventions, we analyzed 28 additional methylomes from mice subjected to lifespan-extending conditions, including Prop1df/df dwarfism, calorie restriction or dietary rapamycin. We found that mice treated with these lifespan-extending interventions were significantly younger in epigenetic age than their untreated, wild-type age-matched controls. Conclusions: This study shows that lifespan-extending conditions can slow molecular changes associated with an epigenetic clock in mice livers

Exaggerated CpH methylation in the autism-affected brain.

BackgroundThe etiology of autism, a complex, heritable, neurodevelopmental disorder, remains largely unexplained. Given the unexplained risk and recent evidence supporting a role for epigenetic mechanisms in the development of autism, we explored the role of CpG and CpH (H = A, C, or T) methylation within the autism-affected cortical brain tissue.MethodsReduced representation bisulfite sequencing (RRBS) was completed, and analysis was carried out in 63 post-mortem cortical brain samples (Brodmann area 19) from 29 autism-affected and 34 control individuals. Analyses to identify single sites that were differentially methylated and to identify any global methylation alterations at either CpG or CpH sites throughout the genome were carried out.ResultsWe report that while no individual site or region of methylation was significantly associated with autism after multi-test correction, methylated CpH dinucleotides were markedly enriched in autism-affected brains (~2-fold enrichment at p < 0.05 cutoff, p = 0.002).ConclusionsThese results further implicate epigenetic alterations in pathobiological mechanisms that underlie autism