A biodegradable calcium sulfite nanoreactor for pH triggered gas therapy in combination with chemotherapy.

As a gasotransmitter, endogenous sulfur dioxide (SO2) plays an important role in cardiovascular regulation. In addition, excessive SO2 can react with overexpressed hydrogen peroxide (H2O2) in tumor cells to generate toxic radicals, which can induce severe oxidative damage to tumor cells and result in cell apoptosis. This highlights the potential of SO2 in oncotherapy. However, the limited availability of endogenous H2O2 and uncontrolled release of SO2 gas significantly impede the effectiveness of SO2 gas therapy. To address this challenge, a biodegradable calcium sulfite (CS) nanocarrier loaded with 10-hydroxycamptothecin (HCPT) was developed for tumor pH-triggered SO2 gas therapy in combination with chemotherapy. This nanoreactor could be degraded in an acidic tumor microenvironment to release SO2 gas and the HCPT drug. The released SO2 gas induced serious oxidative damage to tumor cells by depleting glutathione (GSH) and generating toxic radicals through a reaction with intracellular H2O2. Simultaneously, the HCPT drug promoted tumor cell apoptosis through chemotherapy and boosted SO2 gas therapy by elevating the H2O2 level within the tumor cells. Consequently, the combination of SO2 gas therapy and chemotherapy provided a promising approach for effective tumor treatment.

DMAP2: A Pipeline for Analysis of Whole-Genome-Scale DNA Methylation Sequencing Data.

DNA methylation is well-established as a major epigenetic mechanism that can control gene expression and is involved in both normal development and disease. Analysis of high-throughput-sequencing-based DNA methylation data is a step toward understanding the relationship between disease and phenotype. Analysis of CpG methylation at single-base resolution is routinely done by bisulfite sequencing, in which methylated Cs remain as C while unmethylated Cs are converted to U, subsequently seen as T nucleotides. Sequence reads are aligned to the reference genome using mapping tools that accept the C-T ambiguity. Then, various statistical packages are used to identify differences in methylation between (groups of) samples. We have previously developed the Differential Methylation Analysis Pipeline (DMAP) as an efficient, fast, and flexible tool for this work, both for whole-genome bisulfite sequencing (WGBS) and reduced-representation bisulfite sequencing (RRBS). The protocol described here includes a series of scripts that simplify the use of DMAP tools and that can accommodate the wider range of input formats now in use to perform analysis of whole-genome-scale DNA methylation sequencing data in various biological and clinical contexts. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: DMAP2 workflow for whole-genome bisulfite sequencing (WGBS) and reduced-representation bisulfite sequencing (RRBS).

Sodium houttuyfonate induces bacterial lipopolysaccharide shedding to promote macrophage M1 polarization against acute bacterial lung infection.

Sodium houttuyfonate (SH), derived from the widely utilized natural herb Houttuynia cordata, exhibits an effective therapeutic effect on various diseases, including bacterial and fungal infections, especially the respiratory tract infection. Therefore, the anti-microbial mechanisms of SH may be different from the single-target action mechanism of conventional antibiotics, and further research is needed to clarify this. Firstly, we discovered that SH can effectively intervene in mouse lung infections by reducing bacterial load and acute inflammation response related to pneumonia caused by Pseudomonas aeruginosa. Interestingly, our results confirmed that SH has surface activity and can directly induce changes in the cell wall the shedding of surface lipopolysaccharide (LPS). Additionally, we found that SH-induced shedding of LPS can induce M1 polarization of macrophages in the early stage, leading to the production of corresponding polarization effector molecules. Subsequently, we discovered that SH-induced M1 polarization cells can effectively phagocytose and kill bacterial cells. The protein expression results indicated that SH can enhance the expression of M1 polarization pathway of TLR4/MyD88/NF-κB during the initial phase of macrophage and pathogen interaction. In summary, our results imply that SH could directly induce the shedding of P. aeruginosa LPS in a surfactant-like manner. Afterwards, the SH induced abscisic LPS can initiate the TLR4/MyD88/NF-κB immune pathway to trigger the M1 polarization of macrophages, which might intervene the P. aeruginosa-caused acute lung infection at early stage. Based on these findings, we attempted to coin the term "immune feedback eradication mechanism against pathogen of natural product" to describe this potent antimicrobial mechanism of SH.

Beyond the base pairs: comparative genome-wide DNA methylation profiling across sequencing technologies.

Deoxyribonucleic acid (DNA) methylation plays a key role in gene regulation and is critical for development and human disease. Techniques such as whole-genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS) allow DNA methylation analysis at the genome scale, with Illumina NovaSeq 6000 and MGI Tech DNBSEQ-T7 being popular due to their efficiency and affordability. However, detailed comparative studies of their performance are not available. In this study, we constructed 60 WGBS and RRBS libraries for two platforms using different types of clinical samples and generated approximately 2.8 terabases of sequencing data. We systematically compared quality control metrics, genomic coverage, CpG methylation levels, intra- and interplatform correlations, and performance in detecting differentially methylated positions. Our results revealed that the DNBSEQ platform exhibited better raw read quality, although base quality recalibration indicated potential overestimation of base quality. The DNBSEQ platform also showed lower sequencing depth and less coverage uniformity in GC-rich regions than did the NovaSeq platform and tended to enrich methylated regions. Overall, both platforms demonstrated robust intra- and interplatform reproducibility for RRBS and WGBS, with NovaSeq performing better for WGBS, highlighting the importance of considering these factors when selecting a platform for bisulfite sequencing.

Feasibility assessment and underlying mechanisms of metabisulfite pretreatment for enhanced volatile fatty acids production from anaerobic sludge fermentation.

Employing chemical pretreatment for waste activated sludge (WAS) fermentation is crucial to achieving sustainable sludge management. This study investigated the feasibility of metabisulfite (MS) pretreatment for enhancing volatile fatty acids (VFAs) production from WAS. The results show that after 24-h MS pretreatment, the content of soluble organic matter and loosely bound extracellular polymeric substances (LB-EPS), especially proteins, increased significantly. During the fermentation, MS pretreatment under alkaline conditions was more efficient, with VFA peaking on the fifth day, showing a 140 % increase compared to the alkaline control group. Correlation analysis suggests that the dosage of MS, rather than pH, is closely related to the levels of soluble protein, polysaccharides, LB-EPS, and subsequential VFAs production, while alkaline conditions facilitate the dissolution of total organic carbon. Furthermore, sulfite radicals (SO3•-) are attributed to cell inactivation and lysis, while alkaline conditions initially reduce the size of the flocs, further promoting MS for attacking flocs, thereby improving the performance of fermentation. The study also found that MS pretreatment reduced microbial community diversity, enriched hydrolytic and fermentation bacteria (Actinobacteriota and Firmicutes), and suppressed methanogens (Methanobacteriaceae and Methanosaetaceae), making it a safe, viable, and cost-effective chemical agent for sustainable sludge management.

Rapid Oxidative Dissolution of Zerovalent Iron Induced by Sulfite for Efficient Removal of Arsenate and Arsenite: Selective Formation of Scorodite.

In this study, we proposed a moderate oxidation strategy for accelerating the oxidative dissolution of zerovalent iron (ZVI) using sulfite (S(IV)), thereby improving the removal of As(V) and As(III). Results revealed that, in the presence of 2.0 mM S(IV), both As(V) and As(III) were selectively converted into scorodite at pH0 3.0-7.0, while As(III) oxidation and As(V) immobilization were impressed over pH0 8.0-10.0. Batch experiments, radical quenching experiments, and electron spin resonance (ESR) measurements demonstrated that ZVI initially boosted S(IV) activation to generate SO4•-, •OH, and protons, and in turn, ZVI was further oxidized more intensely by these radicals than by oxygen. Concurrently, substantial protons derived from S(IV) oxidation neutralized hydroxyls produced by ZVI oxidation, maintaining an acidic environment conducive to the generation of scorodite rather than iron (hydr)oxides. Characterizations of X-ray diffraction (XRD), Raman, attenuated total reflectance-Fourier transform infrared (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), X-ray absorption fine structure (XAFS), field emission scanning electron microscopy (FESEM), and high-resolution transmission electron microscopy (HRTEM) confirmed that scorodite was formed in situ and then exfoliated from the surface of ZVI, and approximately 75% of ZVI could still be recovered, which contributed to efficient As removal in successive runs and real As-polluted wastewater. The application of S(IV) achieved a balance among ZVI reactivity improvement, As(V)/As(III) removal, and raw material consumption, making it a promising approach for addressing arsenic contamination in wastewater treatment.

Near-infrared and multifunctional fluorescent probe enabled by cyanopyridine cyanine dye for bisulfite recognition and biological imaging.

SO2 derivatives, sulfite/bisulfite, are widely employed in both the food processing and drug synthesis industries. Despite their widespread application, excessive levels of sulfite/bisulfite can negatively impact human health. Most probes for detecting sulfite/bisulfite are restricted by their fluorescence within the visible spectrum range and poor solubility in aqueous solution, which limit their use in food testing and biological imaging. Herein, a near-infrared probe comprising of the cyanopyridine cyanine skeleton, 4-((Z)-2-((E)-2-chloro-3-(2-cyano-2-(1-methylpyridine-4(1H)-ylidene)ethylidene)cyclohex-1-en-1-yl)-1-cyanovinyl)-1-methylpyridin-1-ium (abbreviated as CCP), was developed. This probe enables precise quantification of bisulfite (HSO3-) in almost pure buffered solutions, showing a near-infrared fluorescence emission at 784 nm with an impressively low detection limit of 0.32 µM. The probe stands out for its exceptional selectivity, minimal susceptibility to interference, and strong adaptability. The probe CCP utilizes the CC bond to trigger a near-infrared fluorescence quenching reaction with HSO3- via nucleophilic addition, which effectively disrupts the large delocalization within the molecule for accurate HSO3- identification. Moreover, the probe has been successfully applied in detecting HSO3- in various food products and living cells, simplifying the measurement of HSO3- content in water samples. This advancement not only enhances the analytical capabilities but also contributes to ensuring food safety and environmental protection. ENVIRONMENTAL IMPLICATION: SO2 derivatives including sulfite/bisulfite, serving dual roles as preservatives and antioxidants, have widespread application across various sectors including food preservation, water sanitation, and the pharmaceutical industry. Despite their widespread application, excessive levels of sulfite/bisulfite can affect human health. Developing methods for precisely and sensitively detecting sulfite/bisulfite in food products and biological samples is important for ensuring food safety and environmental protection. Here, a sensitive near-infrared and multifunctional fluorescent probe in a 99.9 % buffered solution, along with water gel encapsulation, has been successfully applied for the detection of bisulfite in food, authentic water samples, and biological cells.

A dual-functional NIR fluorescence probe for detecting hypochlorous acid and bisulfite in biosystem.

BACKGROUND: Bisulfite (HSO3-) serves as a bleaching agent, antioxidant, antimicrobial, and regulator of enzymatic reactions in biosystem. However, abnormal levels of bisulfite can be detrimental to health. Hypochlorous acid (HOCl), which acts as bioactive small molecules, is crucial for maintaining normal biological functions in living organisms. Disruption of its equilibrium can lead to oxidative stress and various diseases. Therefore, it's essential to monitor the fluctuations of HOCl and HSO3- at cellular and in vivo levels to study their physiological and pathological functions. RESULTS: This study constructed a novel NIR bifunctional colorimetric fluorescent probe using thienocoumarin-indanedione structures to identify hypochlorite (ClO-) and bisulfite (HSO3-). By using CSO-IO to recognize HSO3- and HOCl, two distinct products were generated, displaying green and blue fluorescence, respectively. This property effectively allows for the simultaneous dual-functional detection of HSO3- (LOD: 113 nM) and HOCl (LOD: 43 nM). SIGNIFICANCE: In this work, the biocompatible molecule CSO-IO has been effectively designed to detect HOCl/HSO3- in living cells and zebrafish. As a result, the dual-functional fluorescent probe has the potential to be utilized as a molecular tool to detect HSO3- derived compounds and HOCl simultaneously within the complex biological system.

Developing a fluorescent probe containing benzofuranone moiety for imaging sulphite in living hypoxia pulmonary cells.

In this work, a benzofuranone-derived fluorescent probe BFSF was developed for imaging the sulphite level in living hypoxia pulmonary cells. Under the excitation of 510 nm, BFSF showed a strong fluorescence response at 570 nm when reacted with sulphite. In the solution system, the constructed hypercapnia and serious hypercapnia conditions did not affect the fluorescence response. In comparison with the recently reported probes, BFSF suggested the advantages including rapid response, steady signal reporting, high specificity and low cytotoxicity upon living lung cells. Under a normal incubation atmosphere, BFSF realized the imaging of both exogenous and endogenous sulphite in living pulmonary cells. In particular, BFSF achieved imaging the decrease of the sulphite level under severe hypoxia as well as the recovery of the sulphite level with urgent oxygen supplement. With the imaging capability for the sulphite level in living pulmonary cells under hypoxia conditions, BFSF together with the information herein was meaningful for investigating the anaesthesia-related biological indexes.

Degradation of ß-lactam antibiotics by Fe(III)/HSO3- system and their quantitative structure-activity relationship.

ß-lactam antibiotics, extensively used worldwide, pose significant risks to human health and ecological safety due to their accumulation in the environment. Recent studies have demonstrated the efficacy of transition metal-activated sulfite systems, like Fe(Ⅲ)/HSO3-, in removing PPCPs from water. However, research on their capability to degrade ß-lactam antibiotics remains sparse. This paper evaluates the degradation of 14 types of ß-lactam antibiotics in Fe(Ⅲ)/HSO3- system and establishes a QSAR model correlating molecular descriptors with degradation rates using the MLR method. Using cefazolin as a case study, this research predicts degradation pathways through NPA charge and Fukui function analysis, corroborated by UPLC-MS product analysis. The investigation further explores the influence of variables such as HSO3- dosage, substrate concentration, Fe(Ⅲ) dosage, initial pH and the presence of common seen water matrices including humic acid and bicarbonate on the degradation efficiency. Optimal conditions for cefazolin degradation in Fe(Ⅲ)/HSO3- system were determined to be 93.3 µM HSO3-, 8.12 µM Fe(Ⅲ) and an initial pH of 3.61, under which the interaction of Fe(Ⅲ) dosage with initial pH was found to significantly affect the degradation efficiency. This study not only provides a novel degradation approach for ß-lactam antibiotics but also expands the theoretical application horizon of the Fe(Ⅲ)/HSO3- system.

Rapid and on-site detection of bisulfite via a NIR fluorescent probe: A case study on the emission wavelength of probes with different quinolinium as electron-withdrawing groups.

The emission of venenous sulfur dioxide (SO2) and its derivatives from industrial applications such as coking, transportation and food processing has caused great concern about public health and environmental quality. Probes that enable sensitivity and specificity to detect SO2 derivatives play a crucial role in its regulations and finally mitigating its environmental and health impacts, but fluorescent probes that can accurately, rapidly and on-site detect SO2 derivatives in foodstuffs and environmental systems rarely reported. Herein, a near-infrared (NIR) fluorescent probe (ZTX) for the ratiometric response of bisulfite (HSO3-) was designed and synthesized by regulating the structure of high-performance HSO3- fluorescent probe SL previously reported by us based on structural analyses, theoretical calculations and related literature reports. The Michael addition reaction between the electronic-deficient C=C bond and HSO3- destroys ZTX's π-conjugation system and blocks its intramolecular charge transfer (ICT) process, resulting in a significant fading of the fuchsia solution and the bluish-purple fluorescence turned light blue fluorescence. Fluorescent imaging of HSO3- in live animals utilizing ZTX has been demonstrated. The quantitative analysis of HSO3- in food samples using ZTXvia a smartphone has been also successfully implemented. Simultaneously, the ZTX-based test strips were utilized to quantificationally determine HSO3- in environmental water samples by a smartphone. Consequently, probe ZTX could provide a new method to understand the physiopathological roles of HSO3-, evaluate food safety and monitor environment, and is promising for broad applications.

A fluorescent probe for detecting bisulfite/sulfite in lipid droplets and tracking the dynamics of lipid droplets.

Intracellular lipid droplets (LDs) are important organelles regulating intracellular redox processes. Endogenous bisulfite/sulfite (HSO3-/SO32-) is one of the metabolites of thiol metabolism. The variation in HSO3-/SO32- content around LDs is closely related to cellular homeostasis. However, there is currently no effective method to visualize and quantify the dynamic changes in HSO3-/SO32- content around LDs. In this work, a fluorescent probe MC-BEN utilizing a triphenylamine basic framework was developed to selectively recognize HSO3-/SO32- via a nucleophilic addition reaction. The probe exhibits excellent anti-interference capability, short response time, outstanding photostability, and a low fluorescence detection limit (6.1 µM) for HSO3-/SO32- recognition. More interesting, there is a trend of accelerated contact between LDs and lysosomes after MC-BEN targeting LDs and reacting with endogenous/exogenous HSO3-/SO32-, which may provide new ideas for the study of intracellular lysosomal lipophagy.

Analyzing single-cell bisulfite sequencing data with MethSCAn.

Single-cell bisulfite sequencing (scBS) is a technique that enables the assessment of DNA methylation at single-base pair and single-cell resolution. The analysis of large datasets obtained from scBS requires preprocessing to reduce the data size, improve the signal-to-noise ratio and provide interpretability. Typically, this is achieved by dividing the genome into large tiles and averaging the methylation signals within each tile. Here we demonstrate that this coarse-graining approach can lead to signal dilution. We propose improved strategies to identify more informative regions for methylation quantification and a more accurate quantitation method than simple averaging. Our approach enables better discrimination of cell types and other features of interest and reduces the need for large numbers of cells. We also present an approach to detect differentially methylated regions between groups of cells and demonstrate its ability to identify biologically meaningful regions that are associated with genes involved in the core functions of specific cell types. Finally, we present the software tool MethSCAn for scBS data analysis ( https://anders-biostat.github.io/MethSCAn ).

Role of reactive manganese and oxygen species in the KMnO4/Na2SO3 process for purification of algal-rich water and membrane fouling alleviation.

Ultrafiltration (UF) is a highly efficient technique for algal-rich water purification, but it is heavily contaminated due to the complex water characteristics. To solve this problem, potassium permanganate (KMnO4) oxidation enhanced with sodium sulfite (Na2SO3) was proposed as a pretreatment means. The results showed that the end-normalized flux was elevated from 0.10 to 0.91, and the reversible fouling resistance was reduced by 99.95%. The membrane fouling mechanism also changed obviously, without the generation of cake filtration. Regarding the properties of algal-rich water, the zeta potential was decreased from -29.50 to -5.87 mV after KMnO4/Na2SO3 pretreatment, suggesting that the electrostatic repulsion was significantly reduced. Meanwhile, the fluorescent components in algal-rich water were significantly eliminated, and the removal of dissolved organic carbon was increased to 67.46%. In the KMnO4/Na2SO3 process, reactive manganese species (i.e., Mn(V), Mn(III) and MnO2) and reactive oxygen species (i.e., SO4•- and •OH) played major roles in purifying algal-rich water. Specifically, SO4•-, •OH, Mn(V) and Mn(III) could effectively oxidize algal pollutants. Simultaneously, the in-situ adsorption and coagulation of MnO2 could accelerate the formation of flocs by decreasing the electrostatic repulsion between cells, and protect the algal cells from being excessive oxidized. Overall, the KMnO4/Na2SO3 process showed significant potential for membrane fouling alleviation in purifying algal-rich water.

Whole-genome bisulfite sequencing data analysis learning module on Google Cloud Platform.

This study describes the development of a resource module that is part of a learning platform named 'NIGMS Sandbox for Cloud-based Learning' https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox at the beginning of this Supplement. This module is designed to facilitate interactive learning of whole-genome bisulfite sequencing (WGBS) data analysis utilizing cloud-based tools in Google Cloud Platform, such as Cloud Storage, Vertex AI notebooks and Google Batch. WGBS is a powerful technique that can provide comprehensive insights into DNA methylation patterns at single cytosine resolution, essential for understanding epigenetic regulation across the genome. The designed learning module first provides step-by-step tutorials that guide learners through two main stages of WGBS data analysis, preprocessing and the identification of differentially methylated regions. And then, it provides a streamlined workflow and demonstrates how to effectively use it for large datasets given the power of cloud infrastructure. The integration of these interconnected submodules progressively deepens the user's understanding of the WGBS analysis process along with the use of cloud resources. Through this module, we can enhance the accessibility and adoption of cloud computing in epigenomic research, speeding up the advancements in the related field and beyond. This manuscript describes the development of a resource module that is part of a learning platform named ``NIGMS Sandbox for Cloud-based Learning'' https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox [1] at the beginning of this Supplement. This module delivers learning materials on the analysis of bulk and single-cell ATAC-seq data in an interactive format that uses appropriate cloud resources for data access and analyses.

Rockfish: A transformer-based model for accurate 5-methylcytosine prediction from nanopore sequencing.

DNA methylation plays an important role in various biological processes, including cell differentiation, ageing, and cancer development. The most important methylation in mammals is 5-methylcytosine mostly occurring in the context of CpG dinucleotides. Sequencing methods such as whole-genome bisulfite sequencing successfully detect 5-methylcytosine DNA modifications. However, they suffer from the serious drawbacks of short read lengths and might introduce an amplification bias. Here we present Rockfish, a deep learning algorithm that significantly improves read-level 5-methylcytosine detection by using Nanopore sequencing. Rockfish is compared with other methods based on Nanopore sequencing on R9.4.1 and R10.4.1 datasets. There is an increase in the single-base accuracy and the F1 measure of up to 5 percentage points on R.9.4.1 datasets, and up to 0.82 percentage points on R10.4.1 datasets. Moreover, Rockfish shows a high correlation with whole-genome bisulfite sequencing, requires lower read depth, and achieves higher confidence in biologically important regions such as CpG-rich promoters while being computationally efficient. Its superior performance in human and mouse samples highlights its versatility for studying 5-methylcytosine methylation across varied organisms and diseases. Finally, its adaptable architecture ensures compatibility with new versions of pores and chemistry as well as modification types.

Whole-Genome Bisulfite Sequencing Protocol for the Analysis of Genome-Wide DNA Methylation and Hydroxymethylation Patterns at Single-Nucleotide Resolution.

The analysis of genome-wide epigenomic alterations including DNA methylation and hydroxymethylation has become a subject of intensive research for many biological and clinical questions. DNA methylation analysis bears the particular promise to supplement or replace biochemical and imaging-based tests for the next generation of personalized medicine. Whole-genome bisulfite sequencing (WGBS) using next-generation sequencing technologies is currently considered the gold standard for a comprehensive and quantitative analysis of DNA methylation throughout the genome. However, bisulfite conversion does not allow distinguishing between cytosine methylation and hydroxymethylation requiring an additional chemical or enzymatic step to identify hydroxymethylated cytosines. Here, we provide a detailed protocol based on a commercial kit for the preparation of sequencing libraries for the comprehensive whole-genome analysis of DNA methylation and/or hydroxymethylation. The protocol is based on the construction of sequencing libraries from limited amounts of input DNA by ligation of methylated adaptors to the fragmented DNA prior to bisulfite conversion. For analyses requiring a quantitative distinction between 5-methylcytosine and 5-hydroxymethylcytosines levels, an oxidation step is included in the same workflow to perform oxidative bisulfite sequencing (OxBs-Seq). In this case, two sequencing libraries will be generated and sequenced: a classic methylome following bisulfite conversion and analyzing modified cytosines (not distinguishing between methylated and hydroxymethylated cytosines) and a methylome analyzing only methylated cytosines, respectively. Hydroxymethylation levels are deduced from the differences between the two reactions. We also provide a step-by-step description of the data analysis using publicly available bioinformatic tools. The described protocol has been successfully applied to different human and plant samples and yields robust and reproducible results.

Generation of Whole-Genome Bisulfite Sequencing Libraries for Comprehensive DNA Methylome Analysis.

Whole-genome bisulfite sequencing (WGBS) enables the detection of DNA methylation at a single base-pair resolution. The treatment of DNA with sodium bisulfite allows the discrimination of methylated and unmethylated cytosines, but the power of this technology can be limited by the input amounts of DNA and the length of DNA fragments due to DNA damage caused by the desulfonation process. Here, we describe a WGBS library preparation protocol that minimizes the loss and damage of DNA, generating high-quality libraries amplified with fewer polymerase chain reaction (PCR) cycles, and hence data with fewer PCR duplicates, from lower amounts of input material. Briefly, genomic DNA is sheared, end-repaired, 3'-adenylated, and ligated to adaptors with fewer clean-up steps in between, minimizing DNA loss. The adapter-ligated DNA is then treated with sodium bisulfite and amplified with a few PCR cycles to reach the yield needed for sequencing.

Approaches for the Analysis and Interpretation of Whole-Genome Bisulfite Sequencing Data.

DNA methylation is a covalent modification of DNA that plays important roles in processes such as the regulation of gene expression, transcription factor binding, and suppression of transposable elements. The use of whole-genome bisulfite sequencing (WGBS) enables the genome-wide identification and quantification of DNA methylation patterns at single-base resolution and is the gold standard for the analysis of DNA methylation. However, the computational analysis of WGBS data can be particularly challenging, as many computationally intensive steps are required. Here, we outline step-by-step an approach for the analysis and interpretation of WGBS data. First, sequencing reads must be trimmed, quality-checked, and aligned to the genome. Second, DNA methylation levels are estimated at each cytosine position using the aligned sequence reads of the bisulfite-treated DNA. Third, regions of differential cytosine methylation between samples can be identified. Finally, these data need to be visualized and interpreted in the context of the biological question at hand.

Amplicon-Based Bisulfite Conversion-NGS DNA Methylation Analysis Protocol.

DNA methylation is an important epigenetic modification that regulates chromatin structure and the cell-type-specific expression of genes. The association of aberrant DNA methylation with many diseases, as well as the increasing interest in modifying the methylation mark in a directed manner at genomic sites using epigenome editing for research and therapeutic purposes, increases the need for easy and efficient DNA methylation analysis methods. The standard approach to analyze DNA methylation with a single-cytosine resolution is bisulfite conversion of DNA followed by next-generation sequencing (NGS). In this chapter, we describe a robust, powerful, and cost-efficient protocol for the amplification of target regions from bisulfite-converted DNA, followed by a second PCR step to generate libraries for Illumina NGS. In the two consecutive PCR steps, first, barcodes are added to individual amplicons, and in the second PCR, indices and Illumina adapters are added to the samples. Finally, we describe a detailed bioinformatics approach to extract DNA methylation levels of the target regions from the sequencing data. Combining barcodes with indices enables a high level of multiplexing allowing to sequence multiple pooled samples in the same sequencing run. Therefore, this method is a robust, accurate, quantitative, and cheap approach for the readout of DNA methylation patterns at defined genomic regions.