Protocol for generating high-quality genome-scale DNA methylation sequencing data from human cancer biospecimens.

Aberrant DNA methylation is a universal feature of cancer. Here, we present a protocol for generating high-quality genome-scale DNA methylation sequencing data from a variety of human cancer biospecimens including immortalized cell lines, fresh-frozen surgical resections, and formalin-fixed paraffin-embedded tissues. We describe steps for DNA extraction considerations, reduced representation bisulfite sequencing, data processing and quality control, and downstream data analysis and integration. This protocol is also applicable for other human diseases and methylome profiling in other organisms. For complete details on the use and execution of this protocol, please refer to Rodger et al. (2023).1.

An epigenetic signature of advanced colorectal cancer metastasis.

Colorectal cancer (CRC) is a leading cause of morbidity and mortality worldwide. The majority of CRC deaths are caused by tumor metastasis, even following treatment. There is strong evidence for epigenetic changes, such as DNA methylation, accompanying CRC metastasis and poorer patient survival. Earlier detection and a better understanding of molecular drivers for CRC metastasis are of critical clinical importance. Here, we identify a signature of advanced CRC metastasis by performing whole genome-scale DNA methylation and full transcriptome analyses of paired primary cancers and liver metastases from CRC patients. We observed striking methylation differences between primary and metastatic pairs. A subset of loci showed coordinated methylation-expression changes, suggesting these are potentially epigenetic drivers that control the expression of critical genes in the metastatic cascade. The identification of CRC epigenomic markers of metastasis has the potential to enable better outcome prediction and lead to the discovery of new therapeutic targets.

The transposable element-derived transcript of LIN28B has a placental origin and is not specific to tumours.

Transposable elements (TEs) are genetic elements that have evolved as crucial regulators of human development and cancer, functioning as both genes and regulatory elements. When TEs become dysregulated in cancer cells, they can serve as alternate promoters to activate oncogenes, a process known as onco-exaptation. This study aimed to explore the expression and epigenetic regulation of onco-exaptation events in early human developmental tissues. We discovered co-expression of some TEs and oncogenes in human embryonic stem cells and first trimester and term placental tissues. Previous studies identified onco-exaptation events in various cancer types, including an AluJb SINE element-LIN28B interaction in lung cancer cells, and showed that the TE-derived LIN28B transcript is associated with poor patient prognosis in hepatocellular carcinoma. This study further characterized the AluJb-LIN28B transcript and confirmed that its expression is restricted to the placenta. Targeted DNA methylation analysis revealed differential methylation of the two LIN28B promoters between placenta and healthy somatic tissues, indicating that some TE-oncogene interactions are not cancer-specific but arise from the epigenetic reactivation of developmental TE-derived regulatory events. In conclusion, our findings provide evidence that some TE-oncogene interactions are not limited to cancer and may originate from the epigenetic reactivation of TE-derived regulatory events that are involved in early development. These insights broaden our understanding of the role of TEs in gene regulation and suggest the potential importance of targeting TEs in cancer therapy beyond their conventional use as cancer-specific markers.

Strategy for RNA-Seq Experimental Design and Data Analysis.

Ribonucleic acids (RNAs) are fundamental molecules that control regulation and expression of the genome and therefore the function of a cell. Robust analysis and quantification of RNA transcripts hold critical importance in understanding cell function, altered phenotypes in different biological context, for understanding and targeting diseases. The development of RNA-sequencing (RNA-Seq) now provides opportunities to analyze the expression and function of RNA molecules at an unprecedented scale. However, the strategy for RNA-Seq experimental design and data analysis can substantially differ depending on the biological application. The design choice could also have significant impact for downstream results and interpretation of data. Here we describe key critical considerations required for RNA-Seq experimental design and also describe a step-by-step bioinformatics workflow detailing the different steps required for RNA-Seq data analysis. We believe this article will be a valuable guide for designing and analyzing RNA-Seq data to address a wide range of different biological questions.

Innate immune checkpoint inhibitor resistance is associated with melanoma sub-types exhibiting invasive and de-differentiated gene expression signatures.

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.

Dynamic Epigenetic Changes during a Relapse and Recovery Cycle in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

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.

Generating Sequencing-Based DNA Methylation Maps from Low DNA Input Samples.

Reduced representation bisulfite sequencing (RRBS) is a technique used for assessing genome-wide DNA methylation patterns in eukaryotes. RRBS was introduced to focus on CpG-rich regions that are likely to be of most interest for epigenetic regulation, such as gene promoters and enhancer sequence elements (Meissner et al., Nature 454:766-770, 2008). This "reduced representation" lowers the cost of sequencing and also gives increased depth of coverage, facilitating the resolution of more subtle changes in methylation levels. Here, we describe a modified RRBS sequencing (RRBS-seq) library preparation. Our protocol is optimized for generating single base-resolution libraries when low input DNA is a concern (10-100 ng). Our protocol includes steps to optimize library preparation, such as using deparaffinization solution (when formalin-fixed material is used), and a replacement of gel size-selection with sample purification beads. The described protocol can be accomplished in 3 days and has been successfully applied to tissues or cells from different organisms, including formalin-fixed tissues, to yield robust and reproducible results.

The Mouse Papillomavirus Epigenetic Signature Is Characterised by DNA Hypermethylation after Lesion Regression.

Papillomaviruses (PVs) are double-stranded DNA tumour viruses that can infect cutaneous and mucosal epidermis. Human papillomavirus (HPV) types have been linked to the causality of cutaneous squamous cell carcinoma (cSCC); however, HPV DNA is not always detected in the resultant tumour. DNA methylation is an epigenetic change that can contribute to carcinogenesis. We hypothesise that the DNA methylation pattern in cells is altered following PV infection. We tested if DNA methylation was altered by PV infection in the mouse papillomavirus (MmuPV1) model. Immunosuppressed mice were infected with MmuPV1 on cutaneous tail skin. Immunosuppression was withdrawn for some mice, causing lesions to spontaneously regress. Reduced representation bisulphite sequencing was carried out on DNA from the actively infected lesions, visibly regressed lesions, and mock-infected control mice. DNA methylation libraries were generated and analysed for differentially methylated regions throughout the genome. The presence of MmuPV1 sequences was also assessed. We identified 834 predominantly differentially hypermethylated fragments in regressed lesions, and no methylation differences in actively infected lesions. The promoter regions of genes associated with tumorigenicity, including the tumour suppressor protein DAPK1 and mismatch repair proteins MSH6 and PAPD7, were hypermethylated. Viral DNA was detected in active lesions and in some lesions that had regressed. This is the first description of the genome-wide DNA methylation landscape for active and regressed MmuPV1 lesions. We propose that the DNA hypermethylation in the regressed lesions that we report here may increase the susceptibility of cells to ultraviolet-induced cSCC.

Transcriptional Reprogramming and Constitutive PD-L1 Expression in Melanoma Are Associated with Dedifferentiation and Activation of Interferon and Tumour Necrosis Factor Signalling Pathways.

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.

RepExpress: A Novel Pipeline for the Quantification and Characterization of Transposable Element Expression from RNA-seq Data.

Transposable elements (TEs) are key regulators of both development and disease; however, their repetitive nature presents substantial computational challenges to their analysis. Due to a lack of computational tools and suitable analysis frameworks, TE expression is often not quantified at the locus level. Therefore, we have developed RepExpress, a novel pipeline that enables locus-level TE quantification and characterization. RepExpress enables the characterization of TE expression in a genomic context, and is the first tool focusing on the identification of tissue-specific TE-derived and TE-regulated genes. RepExpress identifies expressed TEs overlapping with annotated genomic features and enables tissue-specific profiles of TE-derived genes. TEs that are expressed with no overlap with any known genomic features are characterized by the closest downstream genomic feature enabling identification of novel TE-gene regulatory relationships. RepExpress takes standard RNA-seq data as input and performs genomic alignment optimized for TEs. Our novel pipeline quantifies expression of both TEs and genes using featureCounts and Stringtie, respectively. RepExpress then filters expressed repeats and characterizes their genomic context, enabling the identification of TEs that overlap with genes, or that may be influencing gene expression. Here, we describe RepExpress, and provide a step-by-step protocol detailing its workflow. We also discuss other TE analysis tools and their applicability to addressing different biological questions. © 2021 Wiley Periodicals LLC. Basic Protocol: RepExpress workflow.

Comparison of Global DNA Methylation Patterns in Human Melanoma Tissues and Their Derivative Cell Lines.

DNA methylation is a heritable epigenetic mark that is fundamental to mammalian development. Aberrant DNA methylation is an epigenetic hallmark of cancer cells. Cell lines are a critical in vitro model and very widely used to unravel mechanisms of cancer cell biology. However, limited data are available to assess whether DNA methylation patterns in tissues are retained when cell lines are established. Here, we provide the first genome-scale sequencing-based methylation map of metastatic melanoma tumour tissues and their derivative cell lines. We show that DNA methylation profiles are globally conserved in vitro compared to the tumour tissue of origin. However, we identify sites that are consistently hypermethylated in cell lines compared to their tumour tissue of origin. The genes associated with these common differentially methylated regions are involved in cell metabolism, cell cycle and apoptosis and are also strongly enriched for the H3K27me3 histone mark and PRC2 complex-related genes. Our data indicate that although global methylation patterns are similar between tissues and cell lines, there are site-specific epigenomic differences that could potentially impact gene expression. Our work provides a valuable resource for identifying false positives due to cell culture and for better interpretation of cancer epigenetics studies in the future.

Genome-wide DNA methylation and RNA expression differences correlate with invasiveness in melanoma cell lines.

Aims & objectives: The aim of this study was to investigate the role of DNA methylation in invasiveness in melanoma cells. Materials & methods: The authors carried out genome-wide transcriptome (RNA sequencing) and reduced representation bisulfite sequencing methylome profiling between noninvasive (n = 4) and invasive melanoma cell lines (n = 5). Results: The integration of differentially expressed genes and differentially methylated fragments (DMFs) identified 12 DMFs (two in AVPI1, one in HMG20B, two in BCL3, one in NTSR1, one in SYNJ2, one in ROBO2 and four in HORMAD2) that overlapped with either differentially expressed genes (eight DMFs and six genes) or cis-targets of lncRNAs (five DMFs associated with cis-targets and four differentially expressed lncRNAs). Conclusions: DNA methylation changes are associated with a number of transcriptional differences observed in noninvasive and invasive phenotypes in melanoma.

Extensive Inter-Cyst DNA Methylation Variation in Autosomal Dominant Polycystic Kidney Disease Revealed by Genome Scale Sequencing.

Autosomal dominant polycystic kidney disease (ADPKD) is a heritable disease characterized by bilateral renal enlargement due to the growth of cysts throughout the kidneys. Inheritance of a disease-causing mutation is required to develop ADPKD, which results in end-stage kidney disease and is associated with a high morbidity. The pathology underlying cyst formation is not well understood. To address this, we have previously shown the global methylome is altered in ADPKD tissue, suggesting a role of DNA methylation in disease-state renal tissue. As cysts are believed to arise independently, we hypothesize that DNA methylation changes vary accordingly. Here we further investigate the role of DNA methylation within independent cysts to characterize key intra-individual changes. We demonstrate that fragments within CpG islands and gene bodies harbor the greatest amount of variation across the ADPKD kidney, while intergenic fragments are comparatively stable. A proportion of variably methylated genes were also differentially methylated in ADPKD tissue. Our data provide evidence that individual molecular mechanisms are operating in the development of each cyst.

Reawakening the Developmental Origins of Cancer Through Transposable Elements.

Transposable elements (TEs) have an established role as important regulators of early human development, functioning as tissue-specific genes and regulatory elements. Functional TEs are highly active during early development, and interact with important developmental genes, some of which also function as oncogenes. Dedifferentiation is a hallmark of cancer, and is characterized by genetic and epigenetic changes that enable proliferation, self-renewal and a metabolism reminiscent of embryonic stem cells. There is also compelling evidence suggesting that the path to dedifferentiation in cancer can contribute to invasion and metastasis. TEs are frequently expressed in cancer, and recent work has identified a newly proposed mechanism involving extensive recruitment of TE-derived promoters to drive expression of oncogenes and subsequently promote oncogenesis-a process termed onco-exaptation. However, the mechanism by which this phenomenon occurs, and the extent to which it contributes to oncogenesis remains unknown. Initial hypotheses have proposed that onco-exaptation events are cancer-specific and arise randomly due to the dysregulated and hypomethylated state of cancer cells and abundance of TEs across the genome. However, we suspect that exaptation-like events may not just arise due to chance activation of novel regulatory relationships as proposed previously, but as a result of the reestablishment of early developmental regulatory relationships. Dedifferentiation in cancer is well-documented, along with expression of TEs. The known interactions between TEs and pluripotency factors such as NANOG and OCTt4 during early development, along with the expression of some placental-specific TE-derived transcripts in cancer support a possible link between TEs and dedifferentiation of tumor cells. Thus, we hypothesize that onco-exaptation events can be associated with the epigenetic reawakening of early developmental TEs to regulate expression of oncogenes and promote oncogenesis. We also suspect that activation of these early developmental regulatory TEs may promote dedifferentiation, although at this stage it is hard to predict whether TE activation is one of the initial drivers of dedifferentiation. We expect that developmental TE activation occurs as a result of the establishment of an epigenetic landscape in cancer that resembles that of early development and that developmental TE activation may also enable cancers to exploit early developmental pathways, repurposing them to promote malignancy.

Characterisation of DNA methylation changes in EBF3 and TBC1D16 associated with tumour progression and metastasis in multiple cancer types.

BACKGROUND: Characteristic DNA methylation differences have been identified between primary and metastatic melanomas at EBF3 and/or TBC1D16 gene loci. To further evaluate whether these epigenetic changes may act more generally as drivers of tumour onset and metastasis, we have investigated DNA methylation changes involving EBF3 and TBC1D16 in additional publicly available data of multiple different tumour types. RESULTS: Promoter hypermethylation and gene body hypomethylation of EBF3 were observed in a number of metastatic tumour types, when compared to normal or primary tumour tissues, as well as in tumour vs normal tissues and in a colorectal primary/metastasis pair, although not all tumour samples or primary/metastasis cancer pairs exhibited altered patterns of EBF3 methylation. In addition, hypomethylation of TBC1D16 was observed in multiple tumours, including a breast cancer primary/metastasis pair, and to a lesser degree in melanoma, although again not all tumours or cancer primary/metastasis pairs exhibited altered patterns of methylation. CONCLUSIONS: These findings suggest characteristic DNA methylation changes in EBF3 and TBC1D16 are relatively common tumour-associated epigenetic events in multiple tumour types, which is consistent with a potential role as more general drivers of tumour progression.

DNA methylation and gene expression alterations in zebrafish early-life stages exposed to the antibacterial agent triclosan.

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-ß 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.

Marked Global DNA Hypomethylation Is Associated with Constitutive PD-L1 Expression in Melanoma.

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.

A Guide for Designing and Analyzing RNA-Seq Data.

The identity of a cell or an organism is at least in part defined by its gene expression and therefore analyzing gene expression remains one of the most frequently performed experimental techniques in molecular biology. The development of the RNA-Sequencing (RNA-Seq) method allows an unprecedented opportunity to analyze expression of protein-coding, noncoding RNA and also de novo transcript assembly of a new species or organism. However, the planning and design of RNA-Seq experiments has important implications for addressing the desired biological question and maximizing the value of the data obtained. In addition, RNA-Seq generates a huge volume of data and accurate analysis of this data involves several different steps and choices of tools. This can be challenging and overwhelming, especially for bench scientists. In this chapter, we describe an entire workflow for performing RNA-Seq experiments. We describe critical aspects of wet lab experiments such as RNA isolation, library preparation and the initial design of an experiment. Further, we provide a step-by-step description of the bioinformatics workflow for different steps involved in RNA-Seq data analysis. This includes power calculations, setting up a computational environment, acquisition and processing of publicly available data if desired, quality control measures, preprocessing steps for the raw data, differential expression analysis, and data visualization. We particularly mention important considerations for each step to provide a guide for designing and analyzing RNA-Seq data.

A streamlined method for analysing genome-wide DNA methylation patterns from low amounts of FFPE DNA.

BACKGROUND: Formalin fixed paraffin embedded (FFPE) tumor samples are a major source of DNA from patients in cancer research. However, FFPE is a challenging material to work with due to macromolecular fragmentation and nucleic acid crosslinking. FFPE tissue particularly possesses challenges for methylation analysis and for preparing sequencing-based libraries relying on bisulfite conversion. Successful bisulfite conversion is a key requirement for sequencing-based methylation analysis. METHODS: Here we describe a complete and streamlined workflow for preparing next generation sequencing libraries for methylation analysis from FFPE tissues. This includes, counting cells from FFPE blocks and extracting DNA from FFPE slides, testing bisulfite conversion efficiency with a polymerase chain reaction (PCR) based test, preparing reduced representation bisulfite sequencing libraries and massively parallel sequencing. RESULTS: The main features and advantages of this protocol are: An optimized method for extracting good quality DNA from FFPE tissues. An efficient bisulfite conversion and next generation sequencing library preparation protocol that uses 50 ng DNA from FFPE tissue. Incorporation of a PCR-based test to assess bisulfite conversion efficiency prior to sequencing. CONCLUSIONS: We provide a complete workflow and an integrated protocol for performing DNA methylation analysis at the genome-scale and we believe this will facilitate clinical epigenetic research that involves the use of FFPE tissue.

Comparative assessment of DNA methylation patterns between reduced representation bisulfite sequencing and Sequenom EpiTyper methylation analysis.

AIM: Validation of sequencing-based DNA methylation data is an important step for meaningful translation of findings. However, there has been limited assessment of different platforms to validate methylation data from next generation sequencing. METHODS: We performed a comparative methylation analysis between the genome-wide platform of reduced representation bisulfite sequencing with a targeted, Sequenom EpiTyper platform (four genes were analyzed in 15 cell lines covering 52 CpG sites). RESULTS: We show that the accuracy of validation substantially improves if results from multiple and adjacent CpG sites are combined rather than at single CpG sites. We demonstrate increased read number improves accuracy of reduced representation bisulfite sequencing results. Further, by using series of replicates, we document variation in samples analyzed by Sequenom EpiTyper, indicating the importance of including replicates to increase precision. CONCLUSION: The results reveal potential sources of bias and provide a guideline for refining study design for DNA methylation analysis.