Euan J. Rodger

Comparison of alignment software for genome-wide bisulphite sequence data.

Recent advances in next generation sequencing (NGS) technology now provide the opportunity to rapidly interrogate the methylation status of the genome. However, there are challenges in handling and interpretation of the methylation sequence data because of its large volume and the consequences of bisulphite modification. We sequenced reduced representation human genomes on the Illumina platform and efficiently mapped and visualized the data with different pipelines and software packages. We examined three pipelines for aligning bisulphite converted sequencing reads and compared their performance. We also comment on pre-processing and quality control of Illumina data. This comparison highlights differences in methods for NGS data processing and provides guidance to advance sequence-based methylation data analysis for molecular biologists.

DMAP: differential methylation analysis package for RRBS and WGBS data

MOTIVATION The rapid development of high-throughput sequencing technologies has enabled epigeneticists to quantify DNA methylation on a massive scale. Progressive increase in sequencing capacity present challenges in terms of processing analysis and the interpretation of the large amount of data; investigating differential methylation between genome-scale data from multiple samples highlights this challenge. RESULTS We have developed a differential methylation analysis package (DMAP) to generate coverage-filtered reference methylomes and to identify differentially methylated regions across multiple samples from reduced representation bisulphite sequencing and whole genome bisulphite sequencing experiments. We introduce a novel fragment-based approach for investigating DNA methylation patterns for reduced representation bisulphite sequencing data. Further, DMAP provides the identity of gene and CpG features and distances to the differentially methylated regions in a format that is easily analyzed with limited bioinformatics knowledge. AVAILABILITY AND IMPLEMENTATION The software has been implemented in C and has been written to ensure portability between different platforms. The source code and documentation is freely available (DMAP: as compressed TAR archive folder) from http://biochem.otago.ac.nz/research/databases-software/. Two test datasets are also available for download from the Web site. Test dataset 1 contains reads from chromosome 1 of a patient and a control, which is used for comparative analysis in the current article. Test dataset 2 contains reads from a part of chromosome 21 of three disease and three control samples for testing the operation of DMAP, especially for the analysis of variance. Example commands for the analyses are included.

Technical Considerations for Reduced Representation Bisulfite Sequencing with Multiplexed Libraries

Reduced representation bisulfite sequencing (RRBS), which couples bisulfite conversion and next generation sequencing, is an innovative method that specifically enriches genomic regions with a high density of potential methylation sites and enables investigation of DNA methylation at single-nucleotide resolution. Recent advances in the Illumina DNA sample preparation protocol and sequencing technology have vastly improved sequencing throughput capacity. Although the new Illumina technology is now widely used, the unique challenges associated with multiplexed RRBS libraries on this platform have not been previously described. We have made modifications to the RRBS library preparation protocol to sequence multiplexed libraries on a single flow cell lane of the Illumina HiSeq 2000. Furthermore, our analysis incorporates a bioinformatics pipeline specifically designed to process bisulfite-converted sequencing reads and evaluate the output and quality of the sequencing data generated from the multiplexed libraries. We obtained an average of 42 million paired-end reads per sample for each flow-cell lane, with a high unique mapping efficiency to the reference human genome. Here we provide a roadmap of modifications, strategies, and trouble shooting approaches we implemented to optimize sequencing of multiplexed libraries on an a RRBS background.

Genome-wide DNA methylation map of human neutrophils reveals widespread inter-individual epigenetic variation.

The extent of variation in DNA methylation patterns in healthy individuals is not yet well documented. Identification of inter-individual epigenetic variation is important for understanding phenotypic variation and disease susceptibility. Using neutrophils from a cohort of healthy individuals, we generated base-resolution DNA methylation maps to document inter-individual epigenetic variation. We identified 12851 autosomal inter-individual variably methylated fragments (iVMFs). Gene promoters were the least variable, whereas gene body and upstream regions showed higher variation in DNA methylation. The iVMFs were relatively enriched in repetitive elements compared to non-iVMFs, and were associated with genome regulation and chromatin function elements. Further, variably methylated genes were disproportionately associated with regulation of transcription, responsive function and signal transduction pathways. Transcriptome analysis indicates that iVMF methylation at differentially expressed exons has a positive correlation and local effect on the inclusion of that exon in the mRNA transcript.

Genome-wide methylation sequencing of paired primary and metastatic cell lines identifies common DNA methylation changes and a role for EBF3 as a candidate epigenetic driver of melanoma metastasis

Epigenetic alterations are increasingly implicated in metastasis, whereas very few genetic mutations have been identified as authentic drivers of cancer metastasis. Yet, to date, few studies have identified metastasis-related epigenetic drivers, in part because a framework for identifying driver epigenetic changes in metastasis has not been established. Using reduced representation bisulfite sequencing (RRBS), we mapped genome-wide DNA methylation patterns in three cutaneous primary and metastatic melanoma cell line pairs to identify metastasis-related epigenetic drivers. Globally, metastatic melanoma cell lines were hypomethylated compared to the matched primary melanoma cell lines. Using whole genome RRBS we identified 75 shared (10 hyper- and 65 hypomethylated) differentially methylated fragments (DMFs), which were associated with 68 genes showing significant methylation differences. One gene, Early B Cell Factor 3 (EBF3), exhibited promoter hypermethylation in metastatic cell lines, and was validated with bisulfite sequencing and in two publicly available independent melanoma cohorts (n = 40 and 458 melanomas, respectively). We found that hypermethylation of the EBF3 promoter was associated with increased EBF3 mRNA levels in metastatic melanomas and subsequent inhibition of DNA methylation reduced EBF3 expression. RNAi-mediated knockdown of EBF3 mRNA levels decreased proliferation, migration and invasion in primary and metastatic melanoma cell lines. Overall, we have identified numerous epigenetic changes characterising metastatic melanoma cell lines, including EBF3-induced aggressive phenotypic behaviour with elevated EBF3 expression in metastatic melanoma, suggesting that EBF3 promoter hypermethylation may be a candidate epigenetic driver of metastasis.

Global Demethylation in Loss of Imprinting Subtype of Wilms Tumor

Epigenetic abnormalities at the IGF2/H19 locus play a key role in the onset of Wilms tumor. These tumors can be classified into three molecular subtypes depending on the events occurring at this locus: loss of imprinting (LOI), loss of heterozygosity (LOH), or retention of imprinting (ROI). As IGF2 LOI is a consequence of aberrant methylation, we hypothesized that this subtype of Wilms tumors might display global abnormalities of methylation. We therefore analyzed the methylation status of satellite DNA, as a surrogate for global methylation in 50 Wilms tumor patients. Satellite methylation was quantified by a methylation‐sensitive quantitative PCR. We confirmed hypomethylation of both satellite α (Sat α) and satellite 2 (Sat 2) DNA in Wilms tumor samples compared with normal kidney. In addition, we found that LOI tumors, unlike ROI or LOH ones, showed concordant hypomethylation of both Sat α and Sat 2 DNA. This would suggest that the LOI subtype of Wilms tumor, which unlike other subtypes results from an epimutation, has a global deregulation of methylation mechanisms.

5-hydroxymethylcytosine: a potential therapeutic target in cancer

The ten-eleven translocation enzymes catalyze the conversion of 5-methylcytosine to 5-hydroxymethylcytosine, a distinct epigenetic mark that has an integral role in active demethylation. Genes that regulate the distribution and amount of 5-hydroxymethylcytosine in the genome could be suitable therapeutic targets to correct abnormal methylation in cancer. Here, we present an overview of the role of the 5-hydroxymethylcytosine pathway in human disease and discuss the emergence of innovative techniques that can map the distribution of 5-hydroxymethylcytosine at high resolution. In the context of current epigenetic therapies and by using recent functional studies, we propose plausible mechanisms to target the 5-hydroxymethylcytosine pathway in cancer. As the study of 5-hydroxymethylcytosine is still in its infancy, we provide future perspectives.

Modeling and structural analysis of PA clan serine proteases

BackgroundSerine proteases account for over a third of all known proteolytic enzymes; they are involved in a variety of physiological processes and are classified into clans sharing structural homology. The PA clan of endopeptidases is the most abundant and over two thirds of this clan is comprised of the S1 family of serine proteases, which bear the archetypal trypsin fold and have a catalytic triad in the order Histidine, Aspartate, Serine. These proteases have been studied in depth and many three dimensional structures have been experimentally determined. However, these structures mostly consist of bacterial and animal proteases, with a small number of plant and fungal proteases and as yet no structures have been determined for protozoa or archaea. The core structure and active site geometry of these proteases is of interest for many applications. This study investigated the structural properties of different S1 family serine proteases from a diverse range of taxa using molecular modeling techniques.ResultsOur predicted models from protozoa, archaea, fungi and plants were combined with the experimentally determined structures of 16 S1 family members and used for analysis of the catalytic core. Amino acid sequences were submitted to SWISS-MODEL for homology-based structure prediction or the LOOPP server for threading-based structure prediction. Predicted models were refined using INSIGHT II and SCRWL and validated against experimental structures. Investigation of secondary structures and electrostatic surface potential was performed using MOLMOL. The structural geometry of the catalytic core shows clear deviations between taxa, but the relative positions of the catalytic triad residues were conserved. Some highly conserved residues potentially contributing to the stability of the structural core were identified. Evolutionary divergence was also exhibited by large variation in secondary structure features outside the core, differences in overall amino acid distribution, and unique surface electrostatic potential patterns between species.ConclusionsEncompassing a wide range of taxa, our structural analysis provides an evolutionary perspective on S1 family serine proteases. Focusing on the common core containing the catalytic site of the enzyme, this analysis is beneficial for future molecular modeling strategies and structural analysis of serine protease models.

Sex differences in DNA methylation and expression in zebrafish brain: a test of an extended 'male sex drive' hypothesis.

The sex drive hypothesis predicts that stronger selection on male traits has resulted in masculinization of the genome. Here we test whether such masculinizing effects can be detected at the level of the transcriptome and methylome in the adult zebrafish brain. Although methylation is globally similar, we identified 914 specific differentially methylated CpGs (DMCs) between males and females (435 were hypermethylated and 479 were hypomethylated in males compared to females). These DMCs were prevalent in gene body, intergenic regions and CpG island shores. We also discovered 15 distinct CpG clusters with striking sex-specific DNA methylation differences. In contrast, at transcriptome level, more female-biased genes than male-biased genes were expressed, giving little support for the male sex drive hypothesis. Our study provides genome-wide methylome and transcriptome assessment and sheds light on sex-specific epigenetic patterns and in zebrafish for the first time.

Placental Hypomethylation Is More Pronounced in Genomic Loci Devoid of Retroelements

The human placenta is hypomethylated compared to somatic tissues. However, the degree and specificity of placental hypomethylation across the genome is unclear. We assessed genome-wide methylation of the human placenta and compared it to that of the neutrophil, a representative homogeneous somatic cell. We observed global hypomethylation in placenta (relative reduction of 22%) compared to neutrophils. Placental hypomethylation was pronounced in intergenic regions and gene bodies, while the unmethylated state of the promoter remained conserved in both tissues. For every class of repeat elements, the placenta showed lower methylation but the degree of hypomethylation differed substantially between these classes. However, some retroelements, especially the evolutionarily younger Alu elements, retained high levels of placental methylation. Surprisingly, nonretrotransposon-containing sequences showed a greater degree of placental hypomethylation than retrotransposons in every genomic element (intergenic, introns, and exons) except promoters. The differentially methylated fragments (DMFs) in placenta and neutrophils were enriched in gene-poor and CpG-poor regions. The placentally hypomethylated DMFs were enriched in genomic regions that are usually inactive, whereas hypermethylated DMFs were enriched in active regions. Hypomethylation of the human placenta is not specific to retroelements, indicating that the evolutionary advantages of placental hypomethylation go beyond those provided by expression of retrotransposons and retrogenes.