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.
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.
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.
Notes how a recently emerged plant disease, bacterial canker of kiwifruit (Actinidia deliciosa and A. chinensis), is caused by Pseudomonas syringae pv. actinidiae (PSA). States how the disease was ...first reported in China and Japan in the 1980s, that a severe outbreak of PSA began in Italy in 2008 and has spread to other European countries, and was found in both New Zealand and Chile in 2010. Sequences the genomes of strains from China and Japan (where the genus Actinidia is endemic), Italy, New Zealand and Chile to study the evolution of the pathogen and analyse the transmission of PSA between countries. Source: National Library of New Zealand Te Puna Matauranga o Aotearoa, licensed by the Department of Internal Affairs for re-use under the Creative Commons Attribution 3.0 New Zealand Licence.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex disease with variable severity. Patients experience frequent relapses where symptoms increase in severity, leaving them with a ...marked reduction in quality of life. Previous work has investigated molecular differences between ME/CFS patients and healthy controls, but not the dynamic changes specific to each individual patient. We applied precision medicine here to map genomic changes in two selected ME/CFS patients through a period that contained a relapse recovery cycle. DNA was isolated from two patients and a healthy age/gender matched control at regular intervals and captured the patient relapse in each case. Reduced representation DNA methylation sequencing profiles were obtained spanning the relapse recovery cycle. Both patients showed a significantly larger methylome variability (10–20-fold) through the period of sampling compared with the control. During the relapse, changes in the methylome profiles of the two patients were detected in regulatory-active regions of the genome that were associated, respectively, with 157 and 127 downstream genes, indicating disturbed metabolic, immune and inflammatory functions. Severe health relapses in the ME/CFS patients resulted in functionally important changes in their DNA methylomes that, while differing between the two patients, led to very similar compromised physiology. DNA methylation as a signature of disease variability in ongoing ME/CFS may have practical applications for strategies to decrease relapse frequency.
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.
There is increasing evidence that toxicant exposure can alter DNA methylation profile, one of the main epigenetic mechanisms, particularly during embryogenesis when DNA methylation patterns are being ...established. In order to investigate the effects of the antibacterial agent Triclosan on DNA methylation and its correlation with gene expression, zebrafish embryos were exposed during 7 days post-fertilization (starting at maximum 8-cells stage) to 50 and 100 μg/l, two conditions for which increased sensitivity and acclimation have been respectively reported. Although global DNA methylation was not significantly affected, a total of 171 differentially methylated fragments were identified by Reduced Representation Bisulfite Sequencing. The majority of these fragments were found between the two exposed groups, reflecting dose-dependant specific responses. Gene ontology analysis revealed that pathways involved in TGF-β signaling were enriched in larvae exposed to 50 μg/l, while de novo pyrimidine biosynthesis functions were overrepresented in fish exposed to 100 μg/l. In addition, gene expression analysis revealed a positive correlation between mRNA levels and DNA methylation patterns in introns, together with significant alterations of the transcription of genes involved in nervous system development, transcriptional factors and histone methyltransferases. Overall this work provides evidence that Triclosan alters DNA methylation in zebrafish exposed during embryogenesis as well as related genes expression and proposes concentration specific modes of action. Further studies will investigate the possible long-term consequences of these alterations, i.e. latent defects associated with developmental exposure and transgenerational effects, and the possible implications in terms of fitness and adaptation to environmental pollutants.
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•Triclosan is a common antibacterial agent with potential effects on aquatic organisms.•Zebrafish were exposed during 7 days of development at 50 and 100 μg/l.•TCS had no effect on global DNA methylation but induced 171 differentially methylated fragments.•A positive correlation between mRNA level and DNA methylation exists in introns.•TCS showed specific effects at both tested concentrations.
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.
DNA methylation is a stable epigenetic mechanism that has important roles in the normal function of a cell and therefore also in disease etiology. Accurate measurements of normal and altered DNA ...methylation patterns are important to understand its role in regulating gene expression and cell phenotype. Remarkable progress has been made over the last decade in developing methodologies to investigate DNA methylation. The availability of next-generation sequencing has enabled the profiling of methylation marks at an unprecedented scale. Several methods that were previously used to profile locus-specific methylation have now been upgraded to a genome-wide scale using high-throughput sequencing or array platforms. However, because there are so many techniques available, researchers are faced with the challenge of assessing the potential merits or limitations of each technique and selecting the appropriate method for their analysis. In this review we discuss the strengths and weaknesses of genome-wide and gene-specific analysis tools for interrogating DNA methylation. We particularly focus on the design and analysis strategies involved. This review will provide a guideline for selecting the appropriate methods and tools for large-scale and locus-specific DNA methylation analysis.
Melanoma is a highly aggressive skin cancer, which, although highly immunogenic, frequently escapes the body’s immune defences. Immune checkpoint inhibitors (ICI), such as anti-PD1, anti-PDL1, and ...anti-CTLA4 antibodies lead to reactivation of immune pathways, promoting rejection of melanoma. However, the benefits of ICI therapy remain limited to a relatively small proportion of patients who do not exhibit ICI resistance. Moreover, the precise mechanisms underlying innate and acquired ICI resistance remain unclear. Here, we have investigated differences in melanoma tissues in responder and non-responder patients to anti-PD1 therapy in terms of tumour and immune cell gene-associated signatures. We performed multi-omics investigations on melanoma tumour tissues, which were collected from patients before starting treatment with anti-PD1 immune checkpoint inhibitors. Patients were subsequently categorized into responders and non-responders to anti-PD1 therapy based on RECIST criteria. Multi-omics analyses included RNA-Seq and NanoString analysis. From RNA-Seq data we carried out HLA phenotyping as well as gene enrichment analysis, pathway enrichment analysis and immune cell deconvolution studies. Consistent with previous studies, our data showed that responders to anti-PD1 therapy had higher immune scores (median immune score for responders = 0.1335, median immune score for non-responders = 0.05426, p-value = 0.01, Mann-Whitney U two-tailed exact test) compared to the non-responders. Responder melanomas were more highly enriched with a combination of CD8+ T cells, dendritic cells (p-value = 0.03) and an M1 subtype of macrophages (p-value = 0.001). In addition, melanomas from responder patients exhibited a more differentiated gene expression pattern, with high proliferative- and low invasive-associated gene expression signatures, whereas tumours from non-responders exhibited high invasive- and frequently neural crest-like cell type gene expression signatures. Our findings suggest that non-responder melanomas to anti-PD1 therapy exhibit a de-differentiated gene expression signature, associated with poorer immune cell infiltration, which establishes a gene expression pattern characteristic of innate resistance to anti-PD1 therapy. Improved understanding of tumour-intrinsic gene expression patterns associated with response to anti-PD1 therapy will help to identify predictive biomarkers of ICI response and may help to identify new targets for anticancer treatment, especially with a capacity to function as adjuvants to improve ICI outcomes.
Constitutive expression of the immune checkpoint, PD-L1, inhibits anti-tumor immune responses in cancer, although the factors involved in PD-L1 regulation are poorly understood. Here we show that ...loss of global DNA methylation, particularly in intergenic regions and repeat elements, is associated with constitutive (PD-L1CON), versus inducible (PD-L1IND), PD-L1 expression in melanoma cell lines. We further show this is accompanied by transcriptomic up-regulation. De novo epigenetic regulators (e.g., DNMT3A) are strongly correlated with PD-L1 expression and methylome status. Accordingly, decitabine-mediated inhibition of global methylation in melanoma cells leads to increased PD-L1 expression. Moreover, viral mimicry and immune response genes are highly expressed in lymphocyte-negative plus PD-L1-positive melanomas, versus PD-L1-negative melanomas in The Cancer Genome Atlas (TCGA). In summary, using integrated genomic analysis we identified that global DNA methylation influences PD-L1 expression in melanoma, and hence melanoma's ability to evade anti-tumor immune responses. These results have implications for combining epigenetic therapy with immunotherapy.
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•Global DNA hypomethylation in melanoma promotes constitutive PD-L1 expression•Transcriptomic up-regulation accompanies constitutive PD-L1 expression in melanoma•Constitutive PD-L1 is associated with reduced DNMT3A expression and viral mimicry•DNA methylation inhibitor treatment of melanoma cells increases PD-L1 expression
Genetics; Genomics; Cancer; Transcriptomics
Melanoma is the most aggressive type of skin cancer, with increasing incidence worldwide. Advances in targeted therapy and immunotherapy have improved the survival of melanoma patients experiencing ...recurrent disease, but unfortunately treatment resistance frequently reduces patient survival. Resistance to targeted therapy is associated with transcriptomic changes and has also been shown to be accompanied by increased expression of programmed death ligand 1 (PD-L1), a potent inhibitor of immune response. Intrinsic upregulation of PD-L1 is associated with genome-wide DNA hypomethylation and widespread alterations in gene expression in melanoma cell lines. However, an in-depth analysis of the transcriptomic landscape of melanoma cells with intrinsically upregulated PD-L1 expression is lacking. To determine the transcriptomic landscape of intrinsically upregulated PD-L1 expression in melanoma, we investigated transcriptomes in melanomas with constitutive versus inducible PD-L1 expression (referred to as PD-L1CON and PD-L1IND). RNA-Seq analysis was performed on seven PD-L1CON melanoma cell lines and ten melanoma cell lines with low inducible PD-L1IND expression. We observed that PD-L1CON melanoma cells had a reprogrammed transcriptome with a characteristic pattern of dedifferentiated gene expression, together with active interferon (IFN) and tumour necrosis factor (TNF) signalling pathways. Furthermore, we identified key transcription factors that were also differentially expressed in PD-L1CON versus PD-L1IND melanoma cell lines. Overall, our studies describe transcriptomic reprogramming of melanomas with PD-L1CON expression.
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