Pavlo Lutsik

Comprehensive analysis of DNA methylation data with RnBeads

RnBeads is a software tool for large-scale analysis and interpretation of DNA methylation data, providing a user-friendly analysis workflow that yields detailed hypertext reports (http://rnbeads.mpi-inf.mpg.de/). Supported assays include whole-genome bisulfite sequencing, reduced representation bisulfite sequencing, Infinium microarrays and any other protocol that produces high-resolution DNA methylation data. Notable applications of RnBeads include the analysis of epigenome-wide association studies and epigenetic biomarker discovery in cancer cohorts.

Assisted reproductive technologies do not enhance the variability of DNA methylation imprints in human

Background Assisted reproductive technologies (ART) such as in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) are believed to destabilise genomic imprints. An increased frequency of Beckwith–Wiedemann syndrome in children born after ART has been reported. Other, mostly epidemiological, studies argue against this finding. Objective To examine the effect of ART on the stability of DNA methylation imprints, DNA was extracted from maternal peripheral blood (MPB), umbilical cord blood (UCB) and amnion/chorion tissue (ACT) of 185 phenotypically normal children (77 ICSI, 35 IVF, and 73 spontaneous conceptions). Using bisulfite based technologies 10 differentially methylated regions (DMRs) were analysed, including KvDMR1, H19, SNRPN, MEST, GRB10, DLK1/MEG3 IG-DMR, GNAS NESP55, GNAS NESPas, GNAS XL-alpha-s and GNAS Exon1A. Results Methylation indices (MI) do not reveal any significant differences at nine DMRs among the conception groups in neither MPB, UCB nor in ACT. The only slightly variable DMR was that of MEST. Here the mean MI was higher in UCB and MPB of IVF cases (mean MI±SD: 0.41±0.03 (UCB) and 0.40±0.03 (MPB)) compared to the ICSI (0.38±0.03, p=0.003 (UCB); 0.37±0.04, p=0.0007 (MPB)) or spontaneous cases (0.38±0.03, p=0.003 (UCB); 0.38±0.04, p=0.02 (MPB)). Weak but suggestive correlations between DMRs were, however, found between MPB, UCB and ACT. Conclusion This study supports the notion that children conceived by ART do not show a higher degree of imprint variability and hence do not have an a priori higher risk for imprinting disorders.

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.

Adult monozygotic twins discordant for intra-uterine growth have indistinguishable genome-wide DNA methylation profiles

BackgroundLow birth weight is associated with an increased adult metabolic disease risk. It is widely discussed that poor intra-uterine conditions could induce long-lasting epigenetic modifications, leading to systemic changes in regulation of metabolic genes. To address this, we acquire genome-wide DNA methylation profiles from saliva DNA in a unique cohort of 17 monozygotic monochorionic female twins very discordant for birth weight. We examine if adverse prenatal growth conditions experienced by the smaller co-twins lead to long-lasting DNA methylation changes.ResultsOverall, co-twins show very similar genome-wide DNA methylation profiles. Since observed differences are almost exclusively caused by variable cellular composition, an original marker-based adjustment strategy was developed to eliminate such variation at affected CpGs. Among adjusted and unchanged CpGs 3,153 are differentially methylated between the heavy and light co-twins at nominal significance, of which 45 show sensible absolute mean β-value differences. Deep bisulfite sequencing of eight such loci reveals that differences remain in the range of technical variation, arguing against a reproducible biological effect. Analysis of methylation in repetitive elements using methylation-dependent primer extension assays also indicates no significant intra-pair differences.ConclusionsSevere intra-uterine growth differences observed within these monozygotic twins are not associated with long-lasting DNA methylation differences in cells composing saliva, detectable with up-to-date technologies. Additionally, our results indicate that uneven cell type composition can lead to spurious results and should be addressed in epigenomic studies.

Bi-PROF: Bisulfite profiling of target regions using 454 GS FLX Titanium technology

The use of next generation sequencing has expanded our view on whole mammalian methylome patterns. In particular, it provides a genome-wide insight of local DNA methylation diversity at single nucleotide level and enables the examination of single chromosome sequence sections at a sufficient statistical power. We describe a bisulfite-based sequence profiling pipeline, Bi-PROF, which is based on the 454 GS-FLX Titanium technology that allows to obtain up to one million sequence stretches at single base pair resolution without laborious subcloning. To illustrate the performance of the experimental workflow connected to a bioinformatics program pipeline (BiQ Analyzer HT) we present a test analysis set of 68 different epigenetic marker regions (amplicons) in five individual patient-derived xenograft tissue samples of colorectal cancer and one healthy colon epithelium sample as a control. After the 454 GS-FLX Titanium run, sequence read processing and sample decoding, the obtained alignments are quality controlled and statistically evaluated. Comprehensive methylation pattern interpretation (profiling) assessed by analyzing 102-104 sequence reads per amplicon allows an unprecedented deep view on pattern formation and methylation marker heterogeneity in tissues concerned by complex diseases like cancer.

Epigenome data release: a participant-centered approach to privacy protection.

Large-scale epigenome mapping by the NIH Roadmap Epigenomics Project, the ENCODE Consortium and the International Human Epigenome Consortium (IHEC) produces genome-wide DNA methylation data at one base-pair resolution. We examine how such data can be made open-access while balancing appropriate interpretation and genomic privacy. We propose guidelines for data release that both reduce ambiguity in the interpretation of open-access data and limit immediate access to genetic variation data that are made available through controlled access.

MeDeCom: discovery and quantification of latent components of heterogeneous methylomes

It is important for large-scale epigenomic studies to determine and explore the nature of hidden confounding variation, most importantly cell composition. We developed MeDeCom as a novel reference-free computational framework that allows the decomposition of complex DNA methylomes into latent methylation components and their proportions in each sample. MeDeCom is based on constrained non-negative matrix factorization with a new biologically motivated regularization function. It accurately recovers cell-type-specific latent methylation components and their proportions. MeDeCom is a new unsupervised tool for the exploratory study of the major sources of methylation variation, which should lead to a deeper understanding and better biological interpretation.

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/.

Identification and analysis of methylation call differences between bisulfite microarray and bisulfite sequencing data with statistical learning techniques

Background DNA methylation is an epigenetic modification known to play a prime role in gene silencing and is an important topic in epigenetic research. However, due to technology-dependent errors there are inconsistencies between methylation measurements from different methods [1]. Incorrect methylation calls could result in the discovery of spurious associations between methylation patterns and specific phenotypes in epigenomewide association studies (EWAS). We worked towards assigning a measure of confidence to individual CpGs to down-weigh or exclude positions with inconsistent measurements in such studies. We used methylation measurements from the Infinium HumanMethylation450 microarray (b450K) and whole genome bisulfite sequencing (bWGBS) to evaluate whether locus-specific measurement differences, Δb = b450K − bWGBS, are predictable using statistical learning techniques.

Mitochondrial DNA Variation and Heteroplasmy in Monozygotic Twins Clinically Discordant for Multiple Sclerosis

We examined the debated link between mitochondrial DNA (mtDNA) variation and multiple sclerosis (MS) using 49 monozygotic (MZ) twin pairs clinically discordant for MS, which enables to associate de novo mtDNA variants, skewed heteroplasmy, and mtDNA copy number with MS manifestation. Ultra‐deep sequencing of blood‐derived mtDNA revealed 25 heteroplasmic variants with potentially pathogenic features in 18 pairs. All variants were pair‐specific and had low and/or similar heteroplasmy levels in both cotwins. In one pair, a confirmed pathogenic variant (m.11778G>A, heteroplasmy ∼50%) associated with Leber hereditary optic neuropathy was detected. Detailed diagnostic investigation revealed subclinical MS signs in the prior nondiseased cotwin. Moreover, neither mtDNA deletions nor copy‐number variations were involved. Furthermore, the majority of heteroplasmic variants were shared among MZ twins and exhibited more similar heteroplasmy levels in the same tissue of MZ twins as compared with different tissues of the same individual. Heteroplasmy levels were also more similar within MZ twins compared with nonidentical siblings. Our analysis excludes mtDNA variation as a major driver of the discordant clinical manifestation of MS in MZ twins, and provides valuable insights into the occurrence and distribution of heteroplasmic variants within MZ twins and nonidentical siblings, and across different tissues.