Endonuclease enrichment TAPS for cost-effective genome-wide base-resolution DNA methylation detection

Whole genome base-resolution methylome sequencing allows for the most comprehensive analysis of DNA methylation, however, the considerable sequencing cost often limits its applications. While reduced representation sequencing can be an affordable alternative, over 80% of CpGs in the genome are not covered. Building on our recently developed TET-assisted pyridine borane sequencing (TAPS) method, we here described endonuclease enrichment TAPS (eeTAPS), which utilizes dihydrouracil (DHU)-cleaving endonuclease digestion of TAPS-converted DNA to enrich methylated CpG sites (mCpGs). eeTAPS can accurately detect 87% of mCpGs in the mouse genome with a sequencing depth equivalent to 4× whole genome sequencing. In comparison, reduced representation TAPS (rrTAPS) detected less than 4% of mCpGs with 2.5× sequencing depth. Our results demonstrate eeTAPS to be a new strategy for cost-effective genome-wide methylation analysis at single-CpG resolution that can fill the gap between whole-genome and reduced representation sequencing.     

Methylation-capture and Next-Generation Sequencing of free circulating DNA from human plasma

Background

Free circulating DNA (fcDNA) has many potential clinical applications, due to the non-invasive way in which it is collected. However, because of the low concentration of fcDNA in blood, genome-wide analysis carries many technical challenges that must be overcome before fcDNA studies can reach their full potential. There are currently no definitive standards for fcDNA collection, processing and whole-genome sequencing. We report novel detailed methodology for the capture of high-quality methylated fcDNA, library preparation and downstream genome-wide Next-Generation Sequencing. We also describe the effects of sample storage, processing and scaling on fcDNA recovery and quality.

Results

Use of serum versus plasma, and storage of blood prior to separation resulted in genomic DNA contamination, likely due to leukocyte lysis. Methylated fcDNA fragments were isolated from 5 donors using a methyl-binding protein-based protocol and appear as a discrete band of ~180 bases. This discrete band allows minimal sample loss at the size restriction step in library preparation for Next-Generation Sequencing, allowing for high-quality sequencing from minimal amounts of fcDNA. Following sequencing, we obtained 37×10⁶-86×10⁶ unique mappable reads, representing more than 50% of total mappable reads. The methylation status of 9 genomic regions as determined by DNA capture and sequencing was independently validated by clonal bisulphite sequencing.

Conclusions

Our optimized methods provide high-quality methylated fcDNA suitable for whole-genome sequencing, and allow good library complexity and accurate sequencing, despite using less than half of the recommended minimum input DNA.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-476) contains supplementary material, which is available to authorized users.     

A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements

We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2′deoxycytidine (DAC), where we found a 1–16% decrease in Alu element and 18–60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation.     

GBSA: a comprehensive software for analysing whole genome bisulfite sequencing data

High-throughput sequencing is increasingly being used in combination with bisulfite (BS) assays to study DNA methylation at nucleotide resolution. Although several programmes provide genome-wide alignment of BS-treated reads, the resulting information is not readily interpretable and often requires further bioinformatic steps for meaningful analysis. Current post-alignment BS-sequencing programmes are generally focused on the gene-specific level, a restrictive feature when analysis in the non-coding regions, such as enhancers and intergenic microRNAs, is required. Here, we present Genome Bisulfite Sequencing Analyser (GBSA—http://ctrad-csi.nus.edu.sg/gbsa), a free open-source software capable of analysing whole-genome bisulfite sequencing data with either a gene-centric or gene-independent focus. Through analysis of the largest published data sets to date, we demonstrate GBSA’s features in providing sequencing quality assessment, methylation scoring, functional data management and visualization of genomic methylation at nucleotide resolution. Additionally, we show that GBSA’s output can be easily integrated with other high-throughput sequencing data, such as RNA-Seq or ChIP-seq, to elucidate the role of methylated intergenic regions in gene regulation. In essence, GBSA allows an investigator to explore not only known loci but also all the genomic regions, for which methylation studies could lead to the discovery of new regulatory mechanisms.     

Long-read whole-genome methylation patterning using enzymatic base conversion and nanopore sequencing

Long-read whole-genome sequencing analysis of DNA methylation would provide useful information on the chromosomal context of gene expression regulation. Here we describe the development of a method that improves the read length generated by using the bisulfite-sequencing-based approach. In this method, we combined recently developed enzymatic base conversion, where an unmethylated cytosine (C) should be converted to thymine (T), with nanopore sequencing. After methylation-sensitive base conversion, the sequencing library was constructed using long-range polymerase chain reaction. This type of analysis is possible using a minimum of 1 ng genomic DNA, and an N50 read length of 3.4–7.6 kb is achieved. To analyze the produced data, which contained a substantial number of base mismatches due to sequence conversion and an inaccurate base read of the nanopore sequencing, a new analytical pipeline was constructed. To demonstrate the performance of long-read methylation sequencing, breast cancer cell lines and clinical specimens were subjected to analysis, which revealed the chromosomal methylation context of key cancer-related genes, allele-specific methylated genes, and repetitive or deletion regions. This method should convert the intractable specimens for which the amount of available genomic DNA is limited to the tractable targets.     

Validation of Pooled Whole-Genome Re-Sequencing in Arabidopsis lyrata

Sequencing pooled DNA of multiple individuals from a population instead of sequencing individuals separately has become popular due to its cost-effectiveness and simple wet-lab protocol, although some criticism of this approach remains. Here we validated a protocol for pooled whole-genome re-sequencing (Pool-seq) of Arabidopsis lyrata libraries prepared with low amounts of DNA (1.6 ng per individual). The validation was based on comparing single nucleotide polymorphism (SNP) frequencies obtained by pooling with those obtained by individual-based Genotyping By Sequencing (GBS). Furthermore, we investigated the effect of sample number, sequencing depth per individual and variant caller on population SNP frequency estimates. For Pool-seq data, we compared frequency estimates from two SNP callers, VarScan and Snape; the former employs a frequentist SNP calling approach while the latter uses a Bayesian approach. Results revealed concordance correlation coefficients well above 0.8, confirming that Pool-seq is a valid method for acquiring population-level SNP frequency data. Higher accuracy was achieved by pooling more samples (25 compared to 14) and working with higher sequencing depth (4.1× per individual compared to 1.4× per individual), which increased the concordance correlation coefficient to 0.955. The Bayesian-based SNP caller produced somewhat higher concordance correlation coefficients, particularly at low sequencing depth. We recommend pooling at least 25 individuals combined with sequencing at a depth of 100× to produce satisfactory frequency estimates for common SNPs (minor allele frequency above 0.05).     

DNA Methylation as a Biomarker for Cardiovascular Disease Risk

Background

Elevated serum homocysteine is associated with an increased risk of cardiovascular disease (CVD). This may reflect a reduced systemic remethylation capacity, which would be expected to cause decreased genomic DNA methylation in peripheral blood leukocytes (PBL).

Methodology/Principal Findings

We examined the association between prevalence of CVD (myocardial infarction, stroke) and its predisposing conditions (hypertension, diabetes) and PBL global genomic DNA methylation as represented by ALU and Satellite 2 (AS) repetitive element DNA methylation in 286 participants of the Singapore Chinese Health Study, a population-based prospective investigation of 63,257 men and women aged 45–74 years recruited during 1993–1998. Men exhibited significantly higher global DNA methylation [geometric mean (95% confidence interval (CI)): 159 (143, 178)] than women [133 (121, 147)] (P = 0·01). Global DNA methylation was significantly elevated in men with a history of CVD or its predisposing conditions at baseline (P = 0·03) but not in women (P = 0·53). Fifty-two subjects (22 men, 30 women) who were negative for these CVD/predisposing conditions at baseline acquired one or more of these conditions by the time of their follow-up I interviews, which took place on average about 5·8 years post-enrollment. Global DNA methylation levels of the 22 incident cases in men were intermediate (AS, 177) relative to the 56 male subjects who remained free of CVD/predisposing conditions at follow-up (lowest AS, 132) and the 51 male subjects with a diagnosis of CVD or predisposing conditions reported at baseline (highest AS 184) (P for trend = 0.0008) No such association was observed in women (P = 0.91). Baseline body mass index was positively associated with AS in both men and women (P = 0·007).

Conclusions/Significance

Our findings indicate that elevated, not decreased, PBL DNA methylation is positively associated with prevalence of CVD/predisposing conditions and obesity in Singapore Chinese.     

High density DNA methylation array with single CpG site resolution

We have developed a new generation of genome-wide DNA methylation BeadChip which allows high-throughput methylation profiling of the human genome. The new high density BeadChip can assay over 480K CpG sites and analyze twelve samples in parallel. The innovative content includes coverage of 99% of RefSeq genes with multiple probes per gene, 96% of CpG islands from the UCSC database, CpG island shores and additional content selected from whole-genome bisulfite sequencing data and input from DNA methylation experts. The well-characterized Infinium® Assay is used for analysis of CpG methylation using bisulfite-converted genomic DNA. We applied this technology to analyze DNA methylation in normal and tumor DNA samples and compared results with whole-genome bisulfite sequencing (WGBS) data obtained for the same samples. Highly comparable DNA methylation profiles were generated by the array and sequencing methods (average R² of 0.95). The ability to determine genome-wide methylation patterns will rapidly advance methylation research.     

Epigenetic Basis of Regeneration: Analysis of Genomic DNA Methylation Profiles in the MRL/MpJ Mouse

Epigenetic regulation plays essential role in cell differentiation and dedifferentiation, which are the intrinsic processes involved in regeneration. To investigate the epigenetic basis of regeneration capacity, we choose DNA methylation as one of the most important epigenetic mechanisms and the MRL/MpJ mouse as a model of mammalian regeneration known to exhibit enhanced regeneration response in different organs. We report the comparative analysis of genomic DNA methylation profiles of the MRL/MpJ and the control C57BL/6J mouse. Methylated DNA immunoprecipitation followed by microarray analysis using the Nimblegen ‘3 × 720 K CpG Island Plus RefSeq Promoter’ platform was applied in order to carry out genome-wide DNA methylation profiling covering 20 404 promoter regions. We identified hundreds of hypo- and hypermethylated genes and CpG islands in the heart, liver, and spleen, and 37 of them in the three tissues. Decreased inter-tissue diversification and the shift of DNA methylation balance upstream the genes distinguish the genomic methylation patterns of the MRL/MpJ mouse from the C57BL/6J. Homeobox genes and a number of other genes involved in embryonic morphogenesis are significantly overrepresented among the genes hypomethylated in the MRL/MpJ mouse. These findings indicate that epigenetic patterning might be a likely molecular basis of regeneration capability in the MRL/MpJ mouse.     

Non-referenced genome assembly from epigenomic short-read data

Current computational methods used to analyze changes in DNA methylation and chromatin modification rely on sequenced genomes. Here we describe a pipeline for the detection of these changes from short-read sequence data that does not require a reference genome. Open source software packages were used for sequence assembly, alignment, and measurement of differential enrichment. The method was evaluated by comparing results with reference-based results showing a strong correlation between chromatin modification and gene expression. We then used our de novo sequence assembly to build the DNA methylation profile for the non-referenced Psammomys obesus genome. The pipeline described uses open source software for fast annotation and visualization of unreferenced genomic regions from short-read data.     

Non-referenced genome assembly from epigenomic short-read data

Current computational methods used to analyze changes in DNA methylation and chromatin modification rely on sequenced genomes. Here we describe a pipeline for the detection of these changes from short-read sequence data that does not require a reference genome. Open source software packages were used for sequence assembly, alignment, and measurement of differential enrichment. The method was evaluated by comparing results with reference-based results showing a strong correlation between chromatin modification and gene expression. We then used our de novo sequence assembly to build the DNA methylation profile for the non-referenced Psammomys obesus genome. The pipeline described uses open source software for fast annotation and visualization of unreferenced genomic regions from short-read data.     

Genome-wide DNA methylation analysis in precursor B-cells

DNA methylation is responsible for regulating gene expression and cellular differentiation and for maintaining genomic stability during normal human development. Furthermore, it plays a significant role in the regulation of hematopoiesis. In order to elucidate the influence of DNA methylation during B-cell development, genome-wide DNA methylation status of pro-B, pre-BI, pre-BII, and naïve-B-cells isolated from human umbilical cord blood was determined using the methylated CpG island recovery assay followed by next generation sequencing. On average, 182–200 million sequences were generated for each precursor B-cell subset in 10 biological replicates. An overall decrease in methylation was observed during the transition from pro-B to pre-BI, whereas no differential methylation was observed in the pre-BI to pre-BII transition or in the pre-BII to naïve B-cell transition. Most of the methylated regions were located within intergenic and intronic regions not present in a CpG island context. Putative novel enhancers were identified in these regions that were differentially methylated between pro-B and pre-BI cells. The genome-wide methylation profiles are publically available and may be used to gain a better understanding of the involvement of atypical DNA methylation in the pathogenesis of malignancies associated with precursor B-cells.     

Mapping the Flavor Contributing Traits on "Fengwei Melon" (Cucumis melo L.) Chromosomes Using Parent Resequencing and Super Bulked-Segregant Analysis

We used a next-generation high-throughput sequencing platform to resequence the Xinguowei and Shouxing melon cultivars, the parents of Fengwei melon. We found 84% of the reads (under a coverage rate of “13×”) placed on the reference genome DHL92. There were 2,550,000 single-nucleotide polymorphisms and 140,000 structural variations in the two genomes. We also identified 1,290 polymorphic genes between Xinguowei and Shouxing. We combined specific length amplified fragment sequencing (SLAF-seq) and bulked-segregant analysis (super-BSA) to analyze the two parents and the F₂ extreme phenotypes. This combined method yielded 12,438,270 reads, 46,087 SLAF tags, and 4,480 polymorphic markers (average depth of 161.81×). There were six sweet trait-related regions containing 13 differential SLAF markers, and 23 sour trait-related regions containing 48 differential SLAF markers. We further fine-mapped the sweet trait to the genomic regions on chromosomes 6, 10, 11, and 12. Correspondingly, we mapped the sour trait-related genomic regions to chromosomes 2, 3, 4, 5, 9, and 12. Finally, we positioned nine of the 61 differential markers in the sweet and sour trait candidate regions on the parental genome. These markers corresponded to one sweet and eight sour trait-related genes. Our study provides a basis for marker-assisted breeding of desirable sweet and sour traits in Fengwei melons.     

Assessing structural variation in a personal genome—towards a human reference diploid genome

Background

Characterizing large genomic variants is essential to expanding the research and clinical applications of genome sequencing. While multiple data types and methods are available to detect these structural variants (SVs), they remain less characterized than smaller variants because of SV diversity, complexity, and size. These challenges are exacerbated by the experimental and computational demands of SV analysis. Here, we characterize the SV content of a personal genome with Parliament, a publicly available consensus SV-calling infrastructure that merges multiple data types and SV detection methods.

Results

We demonstrate Parliament’s efficacy via integrated analyses of data from whole-genome array comparative genomic hybridization, short-read next-generation sequencing, long-read (Pacific BioSciences RSII), long-insert (Illumina Nextera), and whole-genome architecture (BioNano Irys) data from the personal genome of a single subject (HS1011). From this genome, Parliament identified 31,007 genomic loci between 100 bp and 1 Mbp that are inconsistent with the hg19 reference assembly. Of these loci, 9,777 are supported as putative SVs by hybrid local assembly, long-read PacBio data, or multi-source heuristics. These SVs span 59 Mbp of the reference genome (1.8%) and include 3,801 events identified only with long-read data. The HS1011 data and complete Parliament infrastructure, including a BAM-to-SV workflow, are available on the cloud-based service DNAnexus.

Conclusions

HS1011 SV analysis reveals the limits and advantages of multiple sequencing technologies, specifically the impact of long-read SV discovery. With the full Parliament infrastructure, the HS1011 data constitute a public resource for novel SV discovery, software calibration, and personal genome structural variation analysis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1479-3) contains supplementary material, which is available to authorized users.     

Chemoenzymatic labeling of DNA methylation patterns for single-molecule epigenetic mapping

DNA methylation, specifically, methylation of cytosine (C) nucleotides at the 5-carbon position (5-mC), is the most studied and significant epigenetic modification. Here we developed a chemoenzymatic procedure to fluorescently label non-methylated cytosines in CpG context, allowing epigenetic profiling of single DNA molecules spanning hundreds of thousands of base pairs. We used a CpG methyltransferase with a synthetic S-adenosyl-l-methionine cofactor analog to transfer an azide to cytosines instead of the natural methyl group. A fluorophore was then clicked onto the DNA, reporting on the amount and position of non-methylated CpGs. We found that labeling efficiency was increased up to 2-fold by the addition of a nucleosidase, presumably by degrading the inactive by-product of the cofactor after labeling, preventing its inhibitory effect. We used the method to determine the decline in global DNA methylation in a chronic lymphocytic leukemia patient and then performed whole-genome methylation mapping of the model plant Arabidopsis thaliana. Our genome maps show high concordance with published bisulfite sequencing methylation maps. Although mapping resolution is limited by optical detection to 500–1000 bp, the labeled DNA molecules produced by this approach are hundreds of thousands of base pairs long, allowing access to long repetitive and structurally variable genomic regions.     

Many chronological aging clocks can be found throughout the epigenome: Implications for quantifying biological aging

Epigenetic alterations are a hallmark of aging and age‐related diseases. Computational models using DNA methylation data can create “epigenetic clocks” which are proposed to reflect “biological” aging. Thus, it is important to understand the relationship between predictive clock sites and aging biology. To do this, we examined over 450,000 methylation sites from 9,699 samples. We found ~20% of the measured genomic cytosines can be used to make many different epigenetic clocks whose age prediction performance surpasses that of telomere length. Of these predictive sites, the average methylation change over a lifetime was small (~1.5%) and these sites were under‐represented in canonical regions of epigenetic regulation. There was only a weak association between “accelerated” epigenetic aging and disease. We also compare tissue‐specific and pan‐tissue clock performance. This is critical to applying clocks both to new sample sets in basic research, as well as understanding if clinically available tissues will be feasible samples to evaluate “epigenetic aging” in unavailable tissues (e.g., brain). Despite the reproducible and accurate age predictions from DNA methylation data, these findings suggest they may have limited utility as currently designed in understanding the molecular biology of aging and may not be suitable as surrogate endpoints in studies of anti‐aging interventions. Purpose‐built clocks for specific tissues age ranges or phenotypes may perform better for their specific purpose. However, if purpose‐built clocks are necessary for meaningful predictions, then the utility of clocks and their application in the field needs to be considered in that context.     

Microbial single-strand annealing proteins enable CRISPR gene-editing tools with improved knock-in efficiencies and reduced off-target effects

Several existing technologies enable short genomic alterations including generating indels and short nucleotide variants, however, engineering more significant genomic changes is more challenging due to reduced efficiency and precision. Here, we developed RecT Editor via Designer-Cas9-Initiated Targeting (REDIT), which leverages phage single-stranded DNA-annealing proteins (SSAP) RecT for mammalian genome engineering. Relative to Cas9-mediated homology-directed repair (HDR), REDIT yielded up to a 5-fold increase of efficiency to insert kilobase-scale exogenous sequences at defined genomic regions. We validated our REDIT approach using different formats and lengths of knock-in templates. We further demonstrated that REDIT tools using Cas9 nickase have efficient gene-editing activities and reduced off-target errors, measured using a combination of targeted sequencing, genome-wide indel, and insertion mapping assays. Our experiments inhibiting repair enzyme activities suggested that REDIT has the potential to overcome limitations of endogenous DNA repair steps. Finally, our REDIT method is applicable across cell types including human stem cells, and is generalizable to different Cas9 enzymes.     

A simple flow cytometry-based assay to study global methylation levels in onion,a non-model species

DNA methylation is an important epigenetic mark and global methylation dynamics regulate plant developmental processes. Even though genome sequencing technologies have made DNA methylation studies easier, it is difficult in non-model species where genome information is not available. Therefore in this study, we developed a simple assay for analysing global methylation levels in plants by washless immunolabelling of unfixed nuclei using flow cytometry. Onion leaf tissue was used as a model system, and mean fluorescence intensity due to anti-5- methyl cytosine (5-mC) antibodies were used as a measure of global methylation levels. Among three nuclear isolation buffers evaluated, the highest nuclear yield with the low background was obtained with LB01. To maintain a balance between high DNA fluorescence value and low coefficient of variation of DNA peaks, 45 min of hydrolysis with 0.2 N hydrochloric acid was used for chromatin denaturation resulting in six-fold increase in 5-mC fluorescence compared to control. This method was used successfully to detect 5-Azacytidine induced DNA hypomethylation in onion leaf tissues.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01047-6.