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While DNA methylation is established as a major regulator of gene expression in the nucleus, the existence of mitochondrial DNA (mtDNA) methylation remains controversial. Here, we characterised the mtDNA methylation landscape in the prefrontal cortex of neurological healthy individuals (n=26) and patients with Parkinson’s disease (n=27), using a combination of whole genome bisulfite sequencing (WGBS) and bisulfite-independent methods. Accurate mtDNA mapping from WGBS data required alignment to an mtDNA reference only, to avoid misalignment to nuclear mitochondrial pseudogenes. Once correctly aligned, WGBS data provided ultra-deep mtDNA coverage (16,723±7,711), and revealed overall very low levels of cytosine methylation. The highest methylation levels (5.49±0.97%) were found on CpG position m.545, located in the heavy-strand promoter 1 region. The m.545 methylation was validated using a combination of methylation-sensitive DNA digestion and quantitative PCR analysis. We detected no association between mtDNA methylation profile and Parkinson’s disease. Interestingly, m.545 methylation correlated with the levels of mtDNA transcripts, suggesting a putative role in regulating mtDNA gene expression. In addition, we propose a robust framework for methylation analysis of mtDNA from WGBS data, which is less prone to false-positive findings due to misalignment of nuclear mitochondrial pseudogene sequences.Graphical abstract of the analyses and main findingsFresh-frozen brain tissue was obtained from the prefrontal cortex (Brodmann area 9) of 53 individuals, comprising 27 patients with idiopathic PD and 26 healthy controls. Tissue from the same samples was used in three different downstream analyses. WGBS was conducted on all 53 samples and the data were analysed using three different alignment strategies. Alignment against an mtDNA reference only was clearly superior as it gave the highest and most even depth of coverage. WGBS analysis revealed that mtDNA harbours very low levels of cytosine methylation, with the exception of the CpG position m.545 within the HSP1 region (lower right inset). The m.545 methylation was confirmed by bisulfite- and sequencing-independent methods, employing methylation-specific MspJI DNA digestion, followed by quantification with qPCR or fluorescent PCR and capillary electrophoresis. Finally, mtDNA transcript levels were determined by RT-qPCR and correlated to m.545 methylation levels, showing a positive association.

Enhancer DNA methylation and expression of MYBPHL was studied in multiple myeloma (MM). By bisulfite genomic sequencing, among the three CpGs inside the MYBPHL enhancer, CpG1 was significantly hypomethylated in MM cell lines (6.7–50.0%) than normal plasma cells (37.5–75.0%) (P = 0.007), which was negatively correlated with qPCR-measured MYBPHL expression. In RPMI-8226 and WL-2 cells, bearing the highest CpG1 methylation, 5-azadC caused enhancer demethylation and expression of MYBPHL. In primary samples, higher CpG1 methylation was associated with lower MYBPHL expression. By luciferase assay, luciferase activity was enhanced by MYBPHL enhancer compared with empty vector control, but reduced by site-directed mutagenesis of each CpG. RNA-seq data of newly diagnosed MM patients showed that MYBPHL expression was associated with t(11;14). MOLP-8 cells carrying t(11;14) express the highest levels of MYBPHL, and its knockdown reduced cellular proliferation and increased cell death. Herein, as a proof-of-concept, our data demonstrated that the MYBPHL enhancer, particularly CpG1, was hypomethylated and associated with increased MYBPHL expression in MM, which was implicated in myelomagenesis.

Recent studies exploring the impact of methylation in tumor evolution suggest that while the methylation status of many of the CpG sites are preserved across distinct lineages, others are altered as the cancer progresses. Since changes in methylation status of a CpG site may be retained in mitosis, they could be used to infer the progression history of a tumor via single-cell lineage tree reconstruction. In this work, we introduce the first principled distance-based computational method,Sgootr, for inferring a tumor’s single-cell methylation lineage tree and jointly identifying lineage-informative CpG sites which harbor changes in methylation status that are retained along the lineage. We applySgootron the single-cell bisulfite-treated whole genome sequencing data of multiregionally-sampled tumor cells from 9 metastatic colorectal cancer patients made available by Bianet al., as well as multiregionally-sampled single-cell reduced-representation bisulfite sequencing data from a glioblastoma patient made available by Chaligneet al.. We demonstrate that the tumor lineages constructed reveal a simple model underlying colorectal tumor progression and metastatic seeding. A comparison ofSgootragainst alternative approaches shows thatSgootrcan construct lineage trees with fewer migration events and more in concordance with the sequential-progression model of tumor evolution, in time a fraction of that used in prior studies. Interestingly, lineage-informative CpG sites identified bySgootrare in inter-CpG island (CGI) regions, as opposed to CGI’s, which have been the main regions of interest in genomic methylation-related analyses.Sgootris implemented as aSnakemakeworkflow, available athttps://github.com/algo-cancer/Sgootr.Abstract Figure

We have designed two mitochondria targetable probes P-Py and P-An by the π-conjugation of polyaromatic hydrocarbons (pyrene vs. anthracene) with 4-dimethylamino pyridinium. They present an amphiphilic property with excellent solubility in the common polar and non-polar solvents. Both of them demonstrated a significant fluorescence response to bisulfite in Tris-HCl buffer solutions (5 mM, pH = 7.4). By a combination of fluorescence, UV–vis, time-resolved emission, 1H NMR, and ESI-MS, their sensing mechanisms have been elaborated to be a Michael addition. Notably, P-Py also exhibits a sensitivity to the viscosity change with a Stokes shift of 140 nm, due to the restriction of C–C bond rotation. By taking advantages of its good water solubility, low toxicity, and high mitochondrial target, the dual responses of P-Py to exogenous SO2 derivatives and viscosity change in mitochondria were explored by confocal fluorescence microscopy.

In this work, we developed a sandwich DNA-immunosensor for quantification of the methylated tumour suppressor gene O-6-methylguanine-DNA methyltransferase (MGMT), which is a potential biomarker for brain tumours and breast cancer. The biosensor is based on aminated reduced graphene oxide electrode, which is achieved by ammonium hydroxide chemisorption and anti-5-methylcytosine (anti-5mC) as a methylation bioreceptor. The target single-strand (ss) MGMT oligonucleotide is first recognised by its hybridisation with complementary DNA to form double-stranded (ds) MGMT, which is then captured by anti-5mC on the electrode surface due to the presence of methylation. Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and Scanning electron microscopy (SEM) techniques were used to characterise the electrode surface. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques were used for electrochemical measurements. Under optimised conditions, the proposed biosensor is able to quantify a linear range of concentrations of the MGMT gene from 50 fM to 100 pM with a limit of detection (LOD) of 12 fM. The sandwich design facilitates the simultaneous recognition and quantification of DNA methylation, and the amination significantly improves the sensitivity of the biosensor. This biosensor is label-, bisulfite- and PCR-free and has a simple design for cost-efficient production. It can also be tailor-made to detect other methylated genes, which makes it a promising detection platform for DNA methylation-related disease diagnosis and prognosis.

Genetic and epigenetic contributions to various diseases and biological processes have been well-recognized. However, simultaneous identification of single-nucleotide variants (SNVs) and DNA methylation levels from traditional bisulfite sequencing data is still challenging. Here, we develop double strand bisulfite sequencing (DSBS) for genome-wide accurate identification of SNVs and DNA methylation simultaneously at a single-base resolution by using one dataset. Locking Watson and Crick strand together by hairpin adapter followed by bisulfite treatment and massive parallel sequencing, DSBS simultaneously sequences the bisulfite-converted Watson and Crick strand in one paired-end read, eliminating the strand bias of bisulfite sequencing data. Mutual correction of read1 and read2 can estimate the amplification and sequencing errors, and enables our developed computational pipeline, DSBS Analyzer (https://github.com/tianguolangzi/DSBS), to accurately identify SNV and DNA methylation. Additionally, using DSBS, we provide a genome-wide hemimethylation landscape in the human cells, and reveal that the density of DNA hemimethylation sites in promoter region and CpG island is lower than that in other genomic regions. The cost-effective new approach, which decodes DNA methylome and genomic variants simultaneously, will facilitate more comprehensive studies on numerous diseases and biological processes driven by both genetic and epigenetic variations.

Bisulfite (BS) conversion, which includes a series of chemical reactions using bisulfite, is a prerequisite to most DNA methylation analysis methods, and thus is an essential step in the associated research process. Unfortunately, BS conversion leads to the degradation or loss of DNA, which can hinder further downstream analysis. In addition, it is well known that incomplete BS conversion is crucial, as it causes an exaggeration of the DNA methylation level, which can adversely affect the results. Therefore, there have been many attempts to measure three key features of BS conversion: BS conversion efficiency, recovery, and degradation level. In this study, a multiplex quantitative real-time PCR system named BisQuE was suggested to simultaneously analyze three important aspects of the conversion step. By adopting cytosine-free PCR primers for two differently sized multicopy regions, the short amplicon and long amplicon were obtained from both the genomic and BS-converted DNA, thus enabling the obtaining of reliable and sensitive results and the calculation of the degradation level of the conversion step. Also, probes for detecting converted/unconverted templates and C-T indicators for inducing the formula were included in this assay to quantify BS-converted DNA in order to compute the conversion efficiency and recovery. Six BS conversion kits (EZ DNA Methylation-Lightning Kit, Premium Bisulfite kit, MethylEdge® Bisulfite Conversion System, EpiJET Bisulfite Conversion Kit, EpiTect Fast DNA Bisulfite Kit, and NEBNext® Enzymatic Methyl-seq Conversion Module) were tested in 20 samples using 50 ng of genomic DNA as an input with the BisQuE. The conversion efficiency, degradation levels, as well as recovery rates of the kits were investigated. A total of 99.61–99.90% conversion efficiency was perceived for five of the kits, while the NEBNext kit showed about 94%. The lowest degradation level was shown by the NEBNext kit, whereas the other kits were quite similar. The recovery rates of the kits were found to be within the range of 18–50%. A Qubit assay was also used to compare the recovery rate of BisQuE.

R-loops are three-stranded nucleic acid structures with essential roles in many nuclear processes. However, their unchecked accumulation is associated with genome instability and is observed in neurodevelopmental diseases and cancers. Genome-wide profiling of R-loops in normal and diseased cells can help identify locations of pathogenic R-loops and advance efforts to attenuate them. We present an antibody-independent R-loop detection strategy, BisMapR, that combines nuclease-based R-loop isolation with non-denaturing bisulfite chemistry to produce genome-wide profiles that retain strand information. BisMapR achieves greater resolution and is faster than existing strand-specific R-loop profiling strategies. In mouse embryonic stem cells, we apply BisMapR to find that gene promoters form R-loops in both directions and uncover a subset of active enhancers that, despite being bidirectionally transcribed, form R-loops exclusively on one strand. BisMapR reveals a previously unnoticed feature of active enhancers and provides a tool to systematically examine their mechanisms in gene expression.

Nitrate aerosol is ubiquitous in the atmosphere. Nitrate in the particulate and aqueous phase can affect various atmospheric chemical processes through its hygroscopicity and photolysis. The impacts of nitrate photolysis on the heterogeneous oxidation of SO2 have been attracting attention. However, the influence of nitrate on heterogeneous aqueous phase formation of atmospheric sulfate aerosol is still not very clear. In this study, the effects of nitrate on aqueous phase oxidation of bisulfite under different conditions were investigated. Results show that nitrate photolysis can promote the oxidation of bisulfite to sulfate, especially in the presence of O2. It is found that pH plays a significant role in the reaction, and ammonium sulfate has significant impacts on the enhancement of aqueous phase sulfate production through regulating the pH of solution. An apparent synergism is found among halogen chemistry, nitrate and its photochemistry and S (IV) aqueous oxidation, especially the oxidation of halide ions by nitrate and its photolysis and by the intermediate products produced by the free radical chain oxidation of S (IV) in acidic solution, leading to the coupling of the redox cycle of halogen with the oxidation of bisulfite, which promotes the continuous aqueous oxidation of bisulfite and the formation of sulfate. In addition, the role of nitrate itself in the aqueous phase oxidation of bisulfite is revealed. These results provide a new insight into the heterogeneous aqueous phase oxidation pathways and mechanisms of SO2 in cloud and fog droplets and haze particles.

We developed a simple and highly-selective method for 5-methylcytosine detection of specific gene sequence based on binary-probe DNA hybridization. The sequence complementary to the target was designed into two probes, and each fragment of binary probes bound to a relatively short sequence of the target, which made it sensitive to the base mismatches introduced by bisulfite treatment. The advantages of a low detection limit of methylation abundance of 0.1% for the fully methylated target and high sensitivity of 10 pM have been proved by the successful design of binary-probe hybridization. The successful design of the binary probes makes it possible to quantify the average methylation levels of five CpG sites. Thirty-two DNA strands containing 5, 4, 3, 2, 1 and 0 CpG sites were successfully analyzed with the same pair of binary probes. The higher the average methylation level of the target was, the higher the degree of the hybridization reaction. Based on the simple construction of the binary-probe hybridization, the developed biosensor exhibited signals proportional to the average methylation level of the vimentin gene and could evaluate the average methylation level of artificial mixtures. Furthermore, the method has been used to detect vimentin methylation in a genomic context with good specificity, which indicated its potential in the pre-diagnosis of methylation related disease.

Methylation sequencing using bisulfite treatment has revolutionized the field of molecular biology for eukaryotic systems, unveiling levels of intricacy in regulation of gene expression in response to different environmental conditions. While bacteria also utilize methylation to regulate gene expression, bisulfite sequencing does not work as well in bacteria as in eukaryotes, because bacteria methylate adenosine instead of cytosine. Therefore, global bacterial methylation patterns cannot be studied using common Illumina sequencers. In this work, we demonstrate 6mA-seq, a method that can be used to identify patterns in bacteria that methylate adenosines at GATC sites. Furthermore, this method was used on Escherichia coli cultured on four different carbon sources to demonstrate different methylation patterns due to carbon utilization. 6mA-seq can increase the speed in which epigenetic research is conducted in bacteria.

The level and the biological significance of mitochondrial DNA (mtDNA) methylation in human cells is a controversial topic. Using long-read third-generation sequencing technology, mtDNA methylation can be detected directly from the sequencing data, which overcomes previously suggested biases, introduced by bisulfite treatment-dependent methods. We investigated mtDNA from whole blood-derived DNA and established a workflow to detect CpG methylation with Nanopolish. In order to obtain native mtDNA, we adjusted a whole-genome sequencing protocol and performed ligation library preparation and Nanopore sequencing. To validate the workflow, 897bp of methylated and unmethylated synthetic DNA samples at different dilution ratios were sequenced and CpG methylation was detected. Interestingly, we observed that reads with higher methylation in the synthetic DNA did not pass Guppy calling, possibly affecting conclusions about DNA methylation in Nanopore sequencing. We detected in all blood-derived samples overall low-level methylation across the mitochondrial genome, with exceptions at certain CpG sites. Our results suggest that Nanopore sequencing is capable of detecting low-level mtDNA methylation. However, further refinement of the bioinformatical pipelines including Guppy failed reads are recommended.

Las alteraciones en la metilación de dinucleótidos CpG en regiones promotoras es uno de los mecanismos epigenéticos implicados en cáncer que tiene uso potencial como biomarcador. Su evaluación a partir de tejidos fijados en formalina y embebidos en parafina (FFPE) representa un gran desafío dadas la degradación parcial, el entrecruzamiento y las bajas cantidades del DNA obtenido. En esta nota técnica describimos un protocolo para el estudio del estado de metilación del promotor distal del proto-oncogén K-RAS a partir de varias muestras obtenidas de dos tejidos FFPE de cáncer colorrectal con antigüedad de 11 años. Se empleó un protocolo de conversión con bisulfito alternativo al usual; se usó una DNA polimerasa modificada y una PCR anidada y se optimizó la secuenciación directa del DNA convertido con bisulfito. Este protocolo podría ser aplicado para determinar estados de metilación en otros genes y tipos de cáncer en tejidos FFPE.

The predominant methodology for DNA methylation analysis relies on the chemical deamination by sodium bisulfite of unmodified cytosine to uracil to permit the differential readout of methylated cytosines. Bisulfite treatment damages the DNA, leading to fragmentation and loss of long-range methylation information. To overcome this limitation of bisulfite-treated DNA, we applied a new enzymatic deamination approach, termed enzymatic methyl-seq (EM-seq), to long-range sequencing technologies. Our methodology, named long-read enzymatic modification sequencing (LR-EM-seq), preserves the integrity of DNA, allowing long-range methylation profiling of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) over multikilobase length of genomic DNA. When applied to known differentially methylated regions (DMRs), LR-EM-seq achieves phasing of >5 kb, resulting in broader and better defined DMRs compared with that previously reported. This result showed the importance of phasing methylation for biologically relevant questions and the applicability of LR-EM-seq for long-range epigenetic analysis at single-molecule and single-nucleotide resolution.

R-loops are three stranded nucleic acid structures with essential roles in many nuclear processes. However, their unchecked accumulation as seen in some neurodevelopmental diseases and cancers and is associated with compromised genome stability. Genome-wide profiling of R-loops in normal cells and their comparison in disease states can help identify precise locations of pathogenic R-loops and advance efforts to attenuate deviant R-loops while preserving biologically important ones. Toward this, we have developed an antibody-independent R-loop detection strategy, BisMapR, that combines nuclease-based R-loop isolation with non-denaturing bisulfite chemistry to produce high-resolution, genome-wide R-loop profiles that retain strand information. Furthermore, BisMapR achieves greater resolution and is faster than existing strand-specific R-loop profiling strategies. We applied BisMapR to reveal discrete R-loop behavior at gene promoters and enhancers. We show that gene promoters exhibiting antisense transcription form R-loops in both directions. and uncover a subset of active enhancers that, despite being bidirectionally transcribed, form R-loops exclusively on one strand. Thus, BisMapR reveals a previously unnoticed feature of active enhancers and provides a tool to systematically examine their mechanisms in gene expression.

Although various methods have been developed for sequencing cytosine modifications, it is still challenging for specific and quantitative sequencing of individual modification at base-resolution. For example, to obtain both true 5-methylcytosine (5mC) and true 5-hydroxymethylcytosine (5hmC) information, the two major epigenetic modifications, it usually requires subtraction of two methods, which increases noise and requires high sequencing depth. Recently, we developed TET-assisted pyridine borane sequencing (TAPS) for bisulfite-free direct sequencing of 5mC and 5hmC. Here we demonstrate that two sister methods, TAPSβ and chemical-assisted pyridine borane sequencing (CAPS), can be effectively used for subtraction-free and specific whole-genome sequencing of 5mC and 5hmC, respectively. We also demonstrate pyridine borane sequencing (PS) for whole-genome profiling of 5-formylcytosine and 5-carboxylcytosine, the further oxidized derivatives of 5mC and 5hmC. This work completes the set of versatile borane reduction chemistry-based methods as a comprehensive toolkit for direct and quantitative sequencing of all four cytosine epigenetic modifications., Specific and quantitative sequencing of cytosine modifications is challenging at base-resolution. Here the authors present TAPSβ and CAPS for subtraction-free whole genome sequencing of 5mC and 5hmC.

The 5-carbon positions on cytosine nucleotides preceding guanines in genomic DNA (CpG) are common targets for DNA methylation (5mC). DNA methylation removal can occur through both active and passive mechanisms. Ten-eleven translocation enzymes (TETs) oxidize 5mC in a stepwise manner to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5mC can also be removed passively through sequential cell divisions in the absence of DNA methylation maintenance. In this chapter, we describe approaches that couple TET-assisted bisulfite (TAB) and oxidative bisulfite (OxBS) conversion to the Illumina MethylationEPIC BeadChIP (EPIC array) and show how these technologies can be used to distinguish active versus passive DNA demethylation. We also describe integrative bioinformatics pipelines to facilitate this analysis.

The methylation index of the LINE-1 promoter is one of the most commonly used markers for assessing the global level of genome methylation in various human cells and tissues. We developed an NGS-based protocol for DNA methylation analysis of the LINE-1 retrotransposon promoter. This approach allows assessment of the DNA methylation index of 19 CpG sites in the LINE-1 promoter that have the highest tissue- or tumor-specific variability. The method provides a DNA methylation profile for analyzing either the methylation index of each CpG site independently or the mean DNA methylation index across the LINE-1 promoter. The results obtained using the developed method corresponded well to the level of methylation assessed using a commercially available kit for DNA pyrosequencing. In addition, our method provides much more information: 1) the DNA methylation profile of a significant part of the LINE-1 promoter and 2) the level of DNA methylation at individual LINE-1 loci in the genome. The method of targeted bisulfite massive parallel sequencing of the human LINE-1 retrotransposon promoter can be used in large-scale studies of the global level of genome methylation in normal human cells or tumors. To accomplish this, we modified the targeted massive parallel sequencing method based on 16S Metagenomic Sequencing Library Preparation protocol (Illumina, USA) by:•Introduction of the stage of bisulfite conversion of DNA.•Development of specific primers for the LINE-1 sequence., Graphical abstract Image, graphical abstract

Simultaneous measurement of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) at the single-nucleotide level can be obtained by combining data from DNA processing methods including traditional bisulfite (BS), oxidative bisulfite (oxBS), or Tet-assisted (TAB) bisulfite conversion. Array-based technologies have been widely used in this task, due to their time and cost efficiency. For methylation studies using BS data, many protocols and related packages have been suggested in the literature to deal with limitations and confounders that arise from array data. In this chapter, we illustrate how the reader can make small adjustments to these protocols to obtain estimates of methylation and hydroxymethylation proportions.

DNA methylation constitutes the most studied epigenetic mechanism, regulating gene expression in several physiological and pathological states. Targeted methylation polymerase chain reaction (PCR)-based analyses are among the most universal and commonly used techniques in research. They can be of use for validating methylation-based biomarkers to include in clinical practice. Optimal execution and interpretation of data is fundamental for achieving accurate and reproducible results. In this chapter we describe the backbone procedures behind targeted methylation analyses: bisulfite conversion and downstream PCR-based techniques, including real-time quantitative methylation-specific PCR (qMSP) and high-resolution melting (HRM) methylation-sensitive analyses. Specifically, we give details about the protocol, discuss the pros and cons of these methodologies, and give practical tips for achieving optimal results and for troubleshooting.

RNA has coevolved with numerous posttranscriptional modifications to sculpt interactions with proteins and other molecules. One of these modifications is 5-methylcytosine (m5C) and mapping the position and quantifying the level in different types of cellular RNAs and tissues is an important objective in the field of epitranscriptomics. Both in plants and animals bisulfite conversion has long been the gold standard for detection of m5C in DNA but it can also be applied to RNA. Here, we detail methods for highly reproducible bisulfite treatment of RNA, efficient locus-specific PCR amplification, detection of candidate sites by sequencing on the Illumina MiSeq platform, and bioinformatic calling of non-converted sites.

The comparison of methylomes from immune cells enables the identification of differentially methylated regions and thereby region-associated gene loci. Those regions can be used to discriminate one immune cell population from the other, as well as help to identify key molecules and major pathways determining the unique phenotypes of immune cell lineages. The combination of bisulfite treatment of genomic DNA and next-generation sequencing provides the basis for studying epigenetic changes in different immune cell populations. Further development of whole-genome bisulfite sequencing resulted in a protocol for sequencing libraries that accept both single- or double-stranded DNA from fixed or nonfixed cells, respectively. Therefore, researchers can include immune cell populations in their methylation studies whose isolation depends on the staining of intracellular molecules.

Whole genome bisulfite sequencing is currently at the forefront of epigenetic analysis, facilitating the nucleotide-level resolution of 5-methylcytosine (5mC) on a genome-wide scale. Specialised software have been developed to accommodate the unique difficulties in aligning such sequencing reads to a given reference, building on the knowledge acquired from model organisms such as human, or Arabidopsis thaliana. As the field of epigenetics expands its purview to non-model plant species, new challenges arise which bring into question the suitability of previously established tools.Herein, nine short-read aligners are evaluated: Bismark, BS-Seeker2, BSMAP, BWA-meth, ERNE-BS5, GEM3, GSNAP, Last, and segemehl. Precision-recall of simulated alignments, in comparison to real sequencing data obtained from three natural accessions, reveals on-balance that BWA-meth and BSMAP are able to make the best use of the data during mapping. The influence of difficult-to-map regions, characterised by deviations in sequencing depth over repeat annotations, is evaluated in terms of the mean absolute deviation of the resulting methylation calls in comparison to a realistic methylome. Downstream methylation analysis is responsive to the handling of multi-mapping reads relative to mapping quality (MAPQ), and potentially susceptible to bias arising from the increased sequence complexity of densely-methylated reads.

Age-associated DNA methylation reflects aspect of biological aging—therefore epigenetic clocks for mice can elucidate how the aging process in this model organism is affected by specific treatments or genetic background. Initially, age-predictors for mice were trained for genome-wide DNA methylation profiles and we have recently described a targeted assay based on pyrosequencing of DNA methylation at only three age-associated genomic regions. Here, we established alternative approaches using droplet digital PCR (ddPCR) and barcoded bisulfite amplicon sequencing (BBA-seq). At individual CG dinucleotides (CpGs) the correlation of DNA methylation with chronological age was slightly higher for pyrosequencing and ddPCR as compared to BBA-seq. On the other hand, BBA-seq revealed that neighboring CpGs tend to be stochastically modified at murine age-associated regions. Furthermore, the binary sequel of methylated and non-methylated CpGs in individual reads can be used for single-read predictions, which may reflect heterogeneity in epigenetic aging. In comparison to C57BL/6 mice the single-read age-predictions using BBA-seq were also accelerated in the shorter-lived DBA/2 mice, and in C57BL/6 mice with a lifespan quantitative trait locus of DBA/2 mice. Taken together, we describe alternative targeted methods for epigenetic age predictions that provide new perspectives for aging-intervention studies in mice.

DNA cytosine methylation is an important epigenomic mark with a wide range of functions across many organisms. Whole genome bisulfite sequencing (WGBS) is the gold standard to interrogate cyto-sine methylation genome-wide. Algorithms used to map WGBS reads often encode the four-base DNA alphabet with three letters by reducing two bases to a common letter. This encoding substantially reduces the entropy of nucleotide frequencies in the resulting reference genome. Within the paradigm of read mapping by first filtering possible candidate alignments, reduced entropy of the reference can increase the required computing effort. We introduce another bisulfite mapping algorithm (abismal), based on the idea of encoding a four-letter DNA sequence as only two letters, one for purines and one for pyrimidines. We show that this encoding has greater specificity when subsequences are selected from reads for filtration. Through the two-letter encoding, the abismal software tool maps reads in less time and using less memory than most WGBS read mapping software tools, while attaining similar accuracy. This allows in silico methylation analysis to be performed in a wider range of computing machines with limited hardware settings.

Background: A number of plasma methylated DNA biomarkers related to colorectal cancer (CRC) have been identified. However, the effect of methylation level in leukocytes on plasma-based methylation test was rarely reported. Methods: Blood samples from 213 individuals including 91 CRC patients were collected and separated into 3.5 mL of plasma and paired leukocyte fractions. DNA were extracted from plasma and leukocytes and bisulfite converted, followed by ColoDefense test that detects methylated SEPT9 (mSEPT9) and methylated SDC2 (mSDC2) simultaneously in a single qPCR reaction. Results: Both mSEPT9 and mSDC2 levels in leukocytes exhibited no significant difference among CRC, benign tumors and healthy controls. However, mSEPT9 and mSDC2 levels in plasma were significantly higher in CRC group than those in other groups. The sensitivities of mSEPT9 and mSDC2 alone for detecting CRC with plasma samples were 75.8% and 60.4% with specificities of 94.7% and 86.8%, respectively. These two markers in combination exhibited an improved sensitivity of 85.7% for CRC detection with a specificity of 86.8%, mostly attributable to increased sensitivity of 81.8% for detecting stage 0-II CRC. AUC values for mSEPT9 and mSDC2 alone were 0.864 (95% CI: 0.798 - 0.929) and 0.796 (95% CI: 0.719 - 0.874), respectively, but improved to 0.972 (95% CI: 0.949 - 0.996) when combined for ColoDefense test. Conclusions: The leukocytes gDNA will not affect the performance of plasma ColoDefense test, and plasma ColoDefense test exhibited high sensitivity and specificity in a validation set, demonstrating its potential as a non-invasive and cost-effective method for CRC early detection.

Background Despite improvements in the genetic and epigenetic analysis of cell-free DNA (cfDNA), there has been limited focus on assessing the preanalytical variables of recovery efficiency following cfDNA extraction and bisulfite modification. Quantification of recovery efficiency after these steps can facilitate quality assurance and improve reliability when comparing serial samples. Methods We developed an exogenous DNA Construct to Evaluate the Recovery Efficiency of cfDNA extraction and BISulfite modification (CEREBIS) after cfDNA extraction and/or subsequent bisulfite modification from plasma. The strategic placement of cytosine bases in the 180 bp CEREBIS enabled PCR amplification of the construct by a single primer set both after plasma DNA extraction and following subsequent bisulfite modification. Results Plasma samples derived from 8 organ transplant donors and 6 serial plasma samples derived from a liver transplant recipient were spiked with a known number of copies of CEREBIS. Recovery of CEREBIS after cfDNA extraction and bisulfite modification was quantified with high analytical accuracy by droplet digital PCR. The use of CEREBIS and quantification of its recovery was useful in identifying problematic extractions. Furthermore, its use was shown to be invaluable towards improving the reliability of the analysis of serial samples. Conclusions CEREBIS can be used as a spike-in control to address the preanalytical variable of recovery efficiency both after cfDNA extraction from plasma and following bisulfite modification. Our approach can be readily implemented and its application may have significant benefits, especially in settings where longitudinal quantification of cfDNA for disease monitoring is necessary.

Klebsiella pneumoniae (K. pneumonia) is an important pathogen that can cause severe hospital-/community-acquired infections. To panoramically analyze K. pneumoniae’s methylation features, we completed the whole genome sequences of 14 K. pneumoniae strains covering various serotypes, multilocus-sequence typings (MLSTs), clonal groups (CG), viscosity/virulence and drug-resistances, and further characterized their methylomes using PacBio-SMRT and bisulfite technologies. We identified 15 methylation motifs (13 6mA and two 5mC motifs), among which eight were novel ones. Their corresponding MTases were further validated. Additionally, we analyzed the genomic distribution of GATC and CCWGG methylation motifs shared by all strains, and identified differential distributive patterns of some hemi/un-methylated GATC motifs tending to locate in the intergenic regions (IGRs). Specifically, we characterized the in vivo methylation kinetics at single-base resolution on a genome-wide scale by simulating the dynamic processes of replication-mediated passive demethylation and MTase-catalyzed re-methylation. The slower methylation-rates of the GATC motifs in the replication origins (oriC) and IGRs suggest an epigenetic mechanism implicated in the regulation of replication-initiation and transcription. Our findings illustrate the first comprehensive dynamic methylome map of K. pneumonia at single base resolution, and provide an efficient means and important reference for a better understanding of epigenetic regulation in bacteria.

The selective detection of sulfite and bisulfite ions with anthracene-based compounds (CN1 and CN2) as chemo-dosimeters is reported using fluorescence changes in DMSO:HEPES 70:30%, v/v buffer solution (pH 7.4). Upon treatment with sulfite and bisulfite ions, the fluorescence of the probes gets enhanced significantly at 424 nm. The detection of these ions occurs through the nucleophilic addition at the vinylic C-atom of the probes, which terminates the photo-induced electron transfer (PET) process and consequently enhances the fluorescence. This detection mechanism is well supported by 1H NMR titration, HR-MS and DFT/TD-DFT calculations. The pseudo-first-order rate constants for the addition sulfite (at pH 7.4) and bisulfite (at pH 5) to CN1 are determined to be 3.22x10-3 and 5.02x10-3 s-1, respectively. The limits of detection for sulfite and bisulfite are found to be 1.85x10-7 and 1.56x10-7 M, respectively. The probe CN1 was successfully applied to the detection of bisulfite ion in real samples.

Background In mammals, modifications to cytosine bases, particularly in cytosine-guanine (CpG) dinucleotide contexts, play a major role in shaping the epigenome. The canonical epigenetic mark is 5-methylcytosine (5mC), but oxidized versions of 5mC, including 5-hydroxymethylcytosine (5hmC), are now known to be important players in epigenomic dynamics. Understanding the functional role of these modifications in gene regulation, normal development, and pathological conditions requires the ability to localize these modifications in genomic DNA. The classical approach for sequencing cytosine modifications has involved differential deamination via the chemical sodium bisulfite; however, bisulfite is destructive, limiting its utility in important biological or clinical settings where detection of low frequency populations is critical. Additionally, bisulfite fails to resolve 5mC from 5hmC. Scope of review To summarize how enzymatic rather than chemical approaches can be leveraged to localize and resolve different cytosine modifications in a non-destructive manner. Major conclusions Nature offers a suite of enzymes with biological roles in cytosine modification in organisms spanning from bacteriophages to mammals. These enzymatic activities include methylation by DNA methyltransferases, oxidation of 5mC by TET family enzymes, hypermodification of 5hmC by glucosyltransferases, and the generation of transition mutations from cytosine to uracil by DNA deaminases. Here, we describe how insights into the natural reactivities of these DNA-modifying enzymes can be leveraged to convert them into powerful biotechnological tools. Application of these enzymes in sequencing can be accomplished by relying on their natural activity, exploiting their ability to discriminate between cytosine modification states, reacting them with functionalized substrate analogs to introduce chemical handles, or engineering the DNA-modifying enzymes to take on new reactivities. We describe how these enzymatic reactions have been combined and permuted to localize DNA modifications with high specificity and without the destructive limitations posed by chemical methods for epigenetic sequencing.

A new pyrene-based chemodosimeter (RS3) has been designed and synthesized, which is found to be a promising fluorescent probe for detection of bisulphite ion in DMSO:Na2HPO4/citric acid buffer of pH 5. 1H NMR titration indicates that nucleophilic addition of HSO3− to vinylic C-atom of RS3 terminates the π-conjugation and consequently enhanced the fluorescence significantly. The sensing mechanism is further confirmed by density functional theory (DFT) and time dependent density functional theory (TD-DFT) methods. The binding constant of the 1:1 RS3-HSO3 adduct is found to be 0.7 × 103 M−1. The probe RS3 exhibits high selectivity towards bisulphite ion with a limit of detection of 0.15 µM, which is lower than the maximum permissible limit of bisulfite ion (1.25 µM) in food products required by the European Union Regulations. Further, the probe RS3 has been used for the determination of HSO3− in real samples such as distilled water, tap water, red wine, jaggery and white sugar and also successfully applied for the bio-imaging of HSO3− in live cells.

Posttranscriptional RNA modifications have recently emerged as essential posttranscriptional regulators of gene expression. Here we present two methods for single nucleotide resolution detection of 5-formylcytosine (f5C) in RNA. The first relies on chemical protection of f5C against bisulfite treatment, the second method is based on chemical reduction of f5C to hm5C. In combination with regular bisulfite treatment of RNA, the methods allow for precise mapping of f5C. The protocol is used for f5C detection in mtDNA-encoded RNA, however, it can be straightforwardly applied for transcriptome-wide analyses.

Background: Oral squamous cell carcinoma (OSCC) is one of the most important types of oral malignancies. DKK gene family members as well as DKK2/4 have critical roles in regulation of Wnt signaling as one of the main determining pathway in oral carcinogenesis. This study aimed to identify promoter methylation status of DKK2/4 genes to provide possible biomarkers for early detection and treatment of OSCC patients. Methods: A case control study was performed on 31 fresh tissues obtained from oral cavity of patients affected by OSCC and 31 fresh corresponding tissues from normal healthy controls in Tehran and, between the years of 2016-2018. Purified DNA from tissue samples was subjected to bisulfite treatment and then methylation specific polymerase chain reaction (MSP-PCR) was carried out on treated DNA samples. Results: DKK4 promoter was methylated in none of OSCC samples while it was methylated in 16.1% of healthy controls. 16.1% of OSCC samples were detected to be semimethylated and 22.6% of healthy normal samples were methylated for DKK2 promoter gene. Meaningful difference was found in DKK4 promoter methylation among OSCC patients and healthy controls. Significant correlation was found between DKK4 promoter methylation and tumor grade. The age of all enrolled samples was demonstrated to have strong effect on promoter methylation of studied genes. Conclusion: Hypomethylation of DKK2 and DKK4 genes in higher grades of OSCC samples may indicate the pivotal role of their expression in tumor cells invasion and progression through modulation of Wnt signaling pathway. Further study required to determine simultaneous expression of those genes and Wnt signaling elements at mRNA and protein levels.

Displacement loops (D-loops) are signature intermediates formed during homologous recombination. Numerous factors regulate D-loop formation and disruption, thereby influencing crucial aspects of DNA repair, including donor choice and the possibility of crossover outcome. While D-loop detection methods exist, it is currently unfeasible to assess the relationship between D-loop editors and D-loop characteristics such as length and position. Here, we developed a novel in vitro assay to characterize the length and position of individual D-loops with near base-pair resolution and deep coverage, while also revealing their distribution in a population. Non-denaturing bisulfite treatment modifies the cytosines on the displaced strand of the D-loop to uracil, leaving a permanent signature for the displaced strand. Subsequent single-molecule real-time sequencing uncovers the cytosine conversion patch as a D-loop footprint. The D-loop Mapping Assay is widely applicable with different substrates and donor types and can be used to study factors that influence D-loop properties.

Advanced paternal age at fertilization has been suggested to be a risk factor for neurodevelopmental, psychiatric and other disorders in offspring. One emerging hypothesis suggests that altered offspring phenotype is linked with age-related accumulation of epigenetic changes in the sperm of fathers. Given that paternal age is increasing in the developed world, understanding aging-related epigenetic changes in sperm is needed as well as environmental factors that modify such changes. In this study, we characterize age-dependent changes in sperm DNA methylation profiles between young pubertal (postnatal day (PNDs) 65) and mature (PND120) Wistar rats. We also analyze these changes in rats exposed perinatally to 0.2 mg/kg of ubiquitous environmental xenobiotic 2,2’,4,4’-tetrabromodiphenyl ether (BDE-47). Reduced representation bisulfite sequencing (RRBS) libraries were prepared from caudal epididymal sperm DNA and differentially methylated regions (DMRs; ≥ 10x coverage depth, ≥ 3 CpGs per cluster, ≥ 5% methylation change, q < 0.05) were identified via MethPipe package. In control animals, 5,319 age-dependent DMRs were identified, with 99.3% DMRs hypermethylated in mature animals compared to young pubertal rats. These age-related DMRs were enriched for functional categories essential for embryonic development, such as pattern specification, forebrain and sensory organ development, Hippo and Wnt pathways. Age-related changes in sncRNA, reported in different study, target similar list of genes and biological categories.In BDE-47 exposed rats, sperm DNA methylation was higher in young pubertal and lower in mature animals when compared to controls, which resulted in a significant attenuation in the number of age-dependent DMRs (N = 189) identified in the exposed group. In conclusion, our results indicate that the natural aging process has profound effects on sperm methylation levels and this effect may be modified by environmental exposures. Moreover, our results further support the role of epigenetic mechanisms as a likely link betwen paternal age and offspring health and development.

Background The combination of the bisulfite treatment and the next-generation sequencing is an important method for methylation analysis, and aligning the bisulfite-treated reads (BS-reads) is the critical step for the downstream applications. As bisulfite treatment reduces the complexity of the sequences, a large portion of BS-reads might be aligned to multiple locations of the reference genome ambiguously, called multireads. These multireads cannot be employed in the downstream applications since they are likely to introduce artifacts. To identify the best mapping location of each multiread, existing Bayesian-based methods calculate the probability of the read at each position by considering how does it overlap with unique mapped reads. However, $$\sim 25$$ ∼ 25 % of multireads are not overlapped with any unique reads, which are unresolvable for existing method. Results Here we propose a novel method (EM-MUL) that not only rescues multireads overlapped with unique reads, but also uses the overall coverage and accurate base-level alignment to resolve multireads that cannot be handled by current methods. We benchmark our method on both simulated datasets and real datasets. Experimental results show that it is able to align more than 80% of multireads to the best mapping position with very high accuracy. Conclusions EM-MUL is an effective method designed to accurately determine the best mapping position of multireads in BS-reads. For the downstream applications, it is useful to improve the methylation resolution on the repetitive regions of genome. EM-MUL is free available at https://github.com/lmylynn/EM-MUL.

DNA methylation affects gene expressing and alters the genomic stability, leading to various cancers. Hepatocellular carcinoma (HCC) is the fifth common cancerous diseases in the world with a high mortality rate. There is great demand for detection of HCC DNA methylation. Herein we demonstrated a magnetic and flexible screen printed electrode. Au nanoflowers were decorated on the working electrode to improve the sensitivity. Methylated DNA was captured by immunomagnetic isolation, which was bisulfite-free and PCR-free. The electrode underwent flexibility test and characterized by SEM and electrochemical measurements. The assay successfully distinguished methylated and unmethylated samples. HCC cell lines and normal cell line were used to verify the effectiveness of the electrode. This work provides a promising method for HCC early diagnosis and prognosis.