Assessment of RainDrop BS-seq as a method for large-scale,targeted bisulfite sequencing

We present a systematic assessment of RainDrop BS-seq, a novel method for large-scale, targeted bisulfite sequencing using microdroplet-based PCR amplification coupled with next-generation sequencing. We compared DNA methylation levels at 498 target loci (1001 PCR amplicons) in human whole blood, osteosarcoma cells and an archived tumor tissue sample. We assessed the ability of RainDrop BS-seq to accurately measure DNA methylation over a range of DNA quantities (from 10 to 1500 ng), both with and without whole-genome amplification (WGA) following bisulfite conversion. DNA methylation profiles generated using at least 100 ng correlated well (median R = 0.92) with those generated on Illumina Infinium HumanMethylation450 BeadChips, currently the platform of choice for epigenome-wide association studies (EWAS). WGA allowed for testing of samples with a starting DNA amount of 10 and 50 ng, although a reduced correlation was observed (median R = 0.79). We conclude that RainDrop BS-seq is suitable for measuring DNA methylation levels using nanogram quantities of DNA, and can be used to study candidate epigenetic biomarker loci in an accurate and high-throughput manner, paving the way for its application to routine clinical diagnostics.     

Whole genome amplification of sodium bisulfite-treated DNA allows the accurate estimate of methylated cytosine density in limited DNA resources

Sodium bisulfite modification-based fine mapping of methylated cytosines represents the gold standard technique for DNA methylation studies. A major problem with this approach, however is that it results in considerable DNA degradation, and large quantities of genomic DNA material are needed if numerous genomic regions are to be profiled. In this study, we examined whether whole genome amplification (WGA) techniques can be applied to sodium bisulfite-treated DNA and whether WGA would bias DNA methylation results. Sodium bisulfite-treated DNA was amplified using a standard WGA method: optimized primer-extension preamplification (PEP) with degenerate primers. Following the PCR of bisulfite-treated DNA, the DNA methylation profiles of specific DNA fragments were assessed using three approaches: (i) direct sequencing of the overall product; (ii) the sequencing of cloned PCR products; and (iii) methylation-sensitive single nucleotide primer extension (MS-SNuPE)--and compared with those obtained from bisulfite-treated DNA not subjected to WGA. Our data indicates that the DNA methylation profiles obtained from WGA of sodium bisulfite-treated DNA are consistent with those obtained from non-WGA DNA. The average difference in methylation percentage calculated from the two sets of template using MS-SNuPE was 4%. If our results are replicated on other genomic loci, WGA may become a useful technique in DNA methylation studies.     

Enhanced Methylation Analysis by Recovery of Unsequenceable Fragments

Bisulfite sequencing is a valuable tool for mapping the position of 5-methylcytosine in the genome at single base resolution. However, the associated chemical treatment causes strand scission, which depletes the number of sequenceable DNA fragments in a library and thus necessitates PCR amplification. The AT-rich nature of the library generated from bisulfite treatment adversely affects this amplification, resulting in the introduction of major biases that can confound methylation analysis. Here, we report a method that enables more accurate methylation analysis, by rebuilding bisulfite-damaged components of a DNA library. This recovery after bisulfite treatment (ReBuilT) approach enables PCR-free bisulfite sequencing from low nanogram quantities of genomic DNA. We apply the ReBuilT method for the first whole methylome analysis of the highly AT-rich genome of Plasmodium berghei. Side-by-side comparison to a commercial protocol involving amplification demonstrates a substantial improvement in uniformity of coverage and reduction of sequence context bias. Our method will be widely applicable for quantitative methylation analysis, even for technically challenging genomes, and where limited sample DNA is available.     

Whole genome amplification of Chelex-extracted DNA from a single mite: a method for studying genetics of the predatory mite Phytoseiulus persimilis

We developed and optimized a method using Chelex DNA extraction followed by whole genome amplification (WGA) to overcome problems conducting molecular genetic studies due to the limited amount of DNA obtainable from individual small organisms such as predatory mites. The DNA from a single mite, Phytoseiulus persimilis Athias-Henrot (Acari: Phytoseiidae), isolated in Chelex suspension was subjected to WGA. More than 1000-fold amplification of the DNA was achieved using as little as 0.03 ng genomic DNA template. The DNA obtained by the WGA was used for polymerase chain reaction followed by direct sequencing. From WGA DNA, nuclear DNA intergenic spacers ITS1 and ITS2 and a mitochondrial DNA 12S marker were tested in three different geographical populations of the predatory mite: California, the Netherlands, and Sicily. We found a total of four different alleles of the 12S in the Sicilian population, but no polymorphism was identified in the ITS marker. The combination of Chelex DNA extraction and WGA is thus shown to be a simple and robust technique for examining molecular markers for multiple loci by using individual mites. We conclude that the methods, Chelex extraction of DNA followed by WGA, provide a large quantity of DNA template that can be used for multiple PCR reactions useful for genetic studies requiring the genotypes of individual mites.     

高通量DNA甲基化数据的处理和分析方法

DNA甲基化作为一种表观遗传学修饰,在调控基因表达、X染色体失活、印记基因等方面都发挥着重要的作用.不同的DNA甲基化的预处理方法结合二代测序产生了大量的高通量甲基化数据,这些数据的存储、处理和分析是当前亟需解决的问题.在本文中,总结了目前存在的三种高通量DNA甲基化检测技术（限制性内切酶法,亲和纯化法,重亚硫酸盐转换法）,以及针对这些技术产生的高通量数据开发的存储、处理和分析工具.另外,还注重介绍了单碱基水平的DNA甲基化检测技术,BS-Seq的测序原理、数据处理流程以及后续的分析工具.     

Extracting DNA from museum bird eggs, and whole genome amplification of archive DNA

We present a comprehensive protocol for extracting DNA from egg membranes and other internal debris recovered from the interior of blown museum bird eggs. A variety of commercially available DNA extraction methods were found to be applicable. DNA sequencing of polymerase chain reaction (PCR) products for a 176‐bp fragment of mitochondrial DNA was successful for most egg samples (> 78%) even though the amount of DNA extracted (mean = 14.71 ± 4.55 ng/µL) was significantly less than that obtained for bird skin samples (mean = 67.88 ± 4.77 ng/µL). For PCR and sequencing of snipe (Gallinago) DNA, we provide eight new primers for the ‘DNA barcode’ region of COI mtDNA. In various combinations, the primers target a range of PCR products sized from 72 bp to the full ‘barcode’ of 751 bp. Not all possible combinations were tested with archive snipe DNA, but we found a significantly better success rate of PCR amplification for a shorter 176‐bp target compared with a larger 288‐bp fragment (67% vs. 39%). Finally, we explored the feasibility of whole genome amplification (WGA) for extending the use of archive DNA in PCR and sequencing applications. Of two WGA approaches, a PCR‐based method was found to be able to amplify whole genomic DNA from archive skins and eggs from museum bird collections. After WGA, significantly more archive egg samples produced visible PCR products on agarose (56.9% before WGA vs. 79.0% after WGA). However, overall sequencing success did not improve significantly (78.8% compared with 83.0%).     

Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging

DNA methylation plays a key role in epigenetic regulation of eukaryotic genomes. Hence the genome-wide distribution of 5-methylcytosine, or the methylome, has been attracting intense attention. In recent years, whole-genome bisulfite sequencing (WGBS) has enabled methylome analysis at single-base resolution. However, WGBS typically requires microgram quantities of DNA as well as global PCR amplification, thereby precluding its application to samples of limited amounts. This is presumably because bisulfite treatment of adaptor-tagged templates, which is inherent to current WGBS methods, leads to substantial DNA fragmentation. To circumvent the bisulfite-induced loss of intact sequencing templates, we conceived an alternative method termed Post-Bisulfite Adaptor Tagging (PBAT) wherein bisulfite treatment precedes adaptor tagging by two rounds of random primer extension. The PBAT method can generate a substantial number of unamplified reads from as little as subnanogram quantities of DNA. It requires only 100 ng of DNA for amplification-free WGBS of mammalian genomes. Thus, the PBAT method will enable various novel applications that would not otherwise be possible, thereby contributing to the rapidly growing field of epigenomics.     

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.     

A test of the efficacy of whole‐genome amplification on DNA obtained from low‐yield samples

Conservation and population genetic studies are sometimes hampered by insufficient quantities of high quality DNA. One potential way to overcome this problem is through the use of whole genome amplification (WGA) kits. We performed rolling circle WGA on DNA obtained from matched hair and tissue samples of North American red squirrels (Tamiasciurus hudsonicus). Following polymerase chain reaction (PCR) at four microsatellite loci, we compared genotyping success for DNA from different source tissues, both pre‐ and post‐WGA. Genotypes obtained with tissue were robust, whether or not DNA had been subjected to WGA. DNA extracted from hair produced results that were largely concordant with matched tissue samples, although amplification success was reduced and some allelic dropout was observed. WGA of hair samples resulted in a low genotyping success rate and an unacceptably high rate of allelic dropout and genotyping error. The problem was not rectified by conducting PCR of WGA hair samples in triplicate. Therefore, we conclude that WGA is only an effective method of enhancing template DNA quantity when the initial sample is from high‐yield material.     

Archived Guthrie blood spots as a novel source for quantitative DNA methylation analysis

Sodium bisulfite treatment followed by PCR and DNA sequencing is widely considered the gold standard for the analysis of DNA methylation patterns. However, this technique generally requires substantial quantities of genomic DNA as starting material and is often associated with degradation of DNA. Here, we assess the feasibility of performing bisulfite sequencing on DNA isolated from 3-mm diameter punches of dried blood Guthrie spots. We demonstrate that it is possible to perform bisulfite sequencing from both freshly prepared and archived dried blood spots, using a combination of high purity DNA extraction and efficient bisulfite conversion. With the number of new technologies available for DNA methylation studies, we have extended this analysis and have successfully used a high-throughput mass spectrometry method for DNA methylation analysis on these samples. This provides a new source of material for epigenetic analysis of birth samples and provides an invaluable reference point to track temporal change in epigenetic profiles possibly linked with health and disease.     

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.     

Single-molecule polymerase chain reaction reduces bias: application to DNA methylation analysis by bisulfite sequencing

The treatment of DNA with bisulfite, which converts C to U but leaves 5-methyl-C unchanged, forms the basis of many analytical techniques for DNA methylation analysis. Many techniques exist for measuring the methylation state of a single CpG but, for analysis of an entire region, cloning and sequencing remains the gold standard. However, biases in polymerase chain reaction (PCR) amplification and in cloning can skew the results. We hypothesized that single-molecule PCR (smPCR) amplification would eliminate the PCR amplification bias because competition between templates that amplify at different efficiencies no longer exists. The amplified products can be sequenced directly, thus eliminating cloning bias. We demonstrated this accurate and unbiased approach by analyzing a sample that was expected to contain a 50:50 ratio of methylated to unmethylated molecules: a region of the X-linked FMR1 gene from a human female cell line. We compared traditional cloning and sequencing to smPCR and sequencing. Sequencing smPCR products gave an expected methylated to unmethylated ratio of 48:52, whereas conventional cloning and sequencing gave a biased ratio of 72:28. Our results show that smPCR sequencing can eliminate both PCR and cloning bias and represents an attractive approach to bisulfite sequencing.     

Detection of Cytosine Methylation in Ancient DNA from Five Native American Populations Using Bisulfite Sequencing

While cytosine methylation has been widely studied in extant populations, relatively few studies have analyzed methylation in ancient DNA. Most existing studies of epigenetic marks in ancient DNA have inferred patterns of methylation in highly degraded samples using post-mortem damage to cytosines as a proxy for cytosine methylation levels. However, this approach limits the inference of methylation compared with direct bisulfite sequencing, the current gold standard for analyzing cytosine methylation at single nucleotide resolution. In this study, we used direct bisulfite sequencing to assess cytosine methylation in ancient DNA from the skeletal remains of 30 Native Americans ranging in age from approximately 230 to 4500 years before present. Unmethylated cytosines were converted to uracils by treatment with sodium bisulfite, bisulfite products of a CpG-rich retrotransposon were pyrosequenced, and C-to-T ratios were quantified for a single CpG position. We found that cytosine methylation is readily recoverable from most samples, given adequate preservation of endogenous nuclear DNA. In addition, our results indicate that the precision of cytosine methylation estimates is inversely correlated with aDNA preservation, such that samples of low DNA concentration show higher variability in measures of percent methylation than samples of high DNA concentration. In particular, samples in this study with a DNA concentration above 0.015 ng/μL generated the most consistent measures of cytosine methylation. This study presents evidence of cytosine methylation in a large collection of ancient human remains, and indicates that it is possible to analyze epigenetic patterns in ancient populations using direct bisulfite sequencing approaches.     

DHPLC-based method for DNA methylation analysis of differential methylated regions from imprinted genes

The bisulfite genomic sequencing method is one of the most widely used techniques for methylation analysis in heterogeneous unbiased PCR, amplifying for both methylated and unmethylated alleles simultaneously. However, it requires labor-intensive and time-consuming cloning and sequencing steps. In the current study, we used a denaturing high-performance liquid chromatography (DHPLC) procedure in a complementary way with the bisulfite genomic sequencing to analyze the methylation of differentially methylated regions (DMRs) of imprinted genes. We showed reliable and reproducible results in distinguishing overall methylation profiles of DMRs regions of human SNRPN, H19, MEST/PEG1, LIT1, IGF2, TSSC5, WT1 antisense, and mouse H19, Mest/Peg1, Igf2R imprinted genes. These DHPLC profiles were in accordance with bisulfite genomic sequencing data and may serve as a type of "fingerprint," revealing the overall methylation status of DMRs associated with sample heterogeneity. We conclude that DHPLC analysis could be used to increase the throughput efficiency of methylation pattern analysis of imprinted genes after the bisulfite conversion of genomic DNA and unbiased PCR amplification.     

DNA methylation analysis by digital bisulfite genomic sequencing and digital MethyLight

Alterations in cytosine-5 DNA methylation are frequently observed in most types of human cancer. Although assays utilizing PCR amplification of bisulfite-converted DNA are widely employed to analyze these DNA methylation alterations, they are generally limited in throughput capacity, detection sensitivity, and or resolution. Digital PCR, in which a DNA sample is analyzed in distributive fashion over multiple reaction chambers, allows for enumeration of discrete template DNA molecules, as well as sequestration of non-specific primer annealing templates into negative chambers, thereby increasing the signal-to-noise ratio in positive chambers. Here, we have applied digital PCR technology to bisulfite-converted DNA for single-molecule high-resolution DNA methylation analysis and for increased sensitivity DNA methylation detection. We developed digital bisulfite genomic DNA sequencing to efficiently determine single-basepair DNA methylation patterns on single-molecule DNA templates without an interim cloning step. We also developed digital MethyLight, which surpasses traditional MethyLight in detection sensitivity and quantitative accuracy for low quantities of DNA. Using digital MethyLight, we identified single-molecule, cancer-specific DNA hypermethylation events in the CpG islands of RUNX3, CLDN5 and FOXE1 present in plasma samples from breast cancer patients.     

Bisulfite Genomic Sequencing-Derived Methylation Profile of theXistGene throughout Early Mouse Development

Differential epigenetic modification by methylation of CpG dinucleotides is a candidate mechanism that may identify the alleles of imprinted genes and result in monoallelic expression of either the maternal or the paternal allele. Determination of the allelic methylation status of imprinted genes in the gametes and during early development is constrained by the limiting quantities of genomic DNA available from these early developmental stages. To circumvent this problem we have used bisulfite genomic sequencing to determine the allelic methylation status of the minimal promoter and a 1-kb region within theXistgene during preimplantation development. We find that the parentalXistalleles are not differentially methylated in these regions. Our findings are discussed in the context of previous conflicting data obtained using methylation-sensitive restriction enzyme digestion followed by PCR amplification to assay for methylation.     

Reference Materials for Calibration of Analytical Biases in Quantification of DNA Methylation

Most contemporary methods for the quantification of DNA methylation employ bisulfite conversion and PCR amplification. However, many reports have indicated that bisulfite-mediated PCR methodologies can result in inaccurate measurements of DNA methylation owing to amplification biases. To calibrate analytical biases in quantification of gene methylation, especially those that arise during PCR, we utilized reference materials that represent exact bisulfite-converted sequences with 0% and 100% methylation status of specific genes. After determining relative quantities using qPCR, pairs of plasmids were gravimetrically mixed to generate working standards with predefined DNA methylation levels at 10% intervals in terms of mole fractions. The working standards were used as controls to optimize the experimental conditions and also as calibration standards in melting-based and sequencing-based analyses of DNA methylation. Use of the reference materials enabled precise characterization and proper calibration of various biases during PCR and subsequent methylation measurement processes, resulting in accurate measurements.     

Differential methylation analysis for bisulfite sequencing using DSS

Bisulfite sequencing (BS-seq) technology measures DNA methylation at single nucleotide resolution. A key task in BS-seq data analysis is to identify differentially methylation (DM) under different conditions. Here we provide a tutorial for BS-seq DM analysis using Bioconductor package DSS. DSS uses a beta-binomial model to characterize the sequence counts from BS-seq, and implements rigorous statistical method for hypothesis testing. It provides flexible functionalities for a variety of DM analyses.     

Preparation of genome-wide DNA fragment libraries using bisulfite in polyacrylamide gel electrophoresis slices with formamide denaturation and quality control for massively parallel sequencing by oligonucleotide ligation and detection

Bisulfite sequencing is widely used for analysis of DNA methylation status (i.e., 5-methylcytosine [5mC] vs. cytosine [C]) in CpG-rich or other loci in genomic DNA (gDNA). Such methods typically involve reaction of gDNA with bisulfite followed by polymerase chain reaction (PCR) amplification of specific regions of interest that, overall, converts C→T (thymine) and 5mC→C and then capillary sequencing to measure C versus T composition at CpG sites. Massively parallel sequencing by oligonucleotide ligation and detection (SOLiD) has recently enabled relatively low-cost whole genome sequencing, and it would be highly desirable to apply such massively parallel sequencing to bisulfite-converted whole genomes to determine DNA methylation status of an entire genome, which has heretofore not been reported. As an initial step toward achieving this goal, we have extended our ongoing interest in improving bisulfite conversion sample preparation to include a human genome-wide fragment library for SOliD. The current article features novel use of formamide denaturant during bisulfite conversion of a suitably constructed library directly in a band slice from polyacryamide gel electrophoresis (PAGE). To validate this new protocol for 5mC-protected fragment library conversion, which we refer to as Bis-PAGE, capillary-based size analysis and Sanger sequencing were carried out for individual amplicons derived from single-molecule PCR (smPCR) of randomly selected library fragments. smPCR/Capillary Sanger sequencing of approximately 200 amplicons unambiguously demonstrated greater than 99% C→T conversion. All of these approximately 200 Sanger sequences were analyzed with a previously published web-accessible bioinformatics tool (methBLAST) for mapping to human chromosomes, the results of which indicated random distribution of analyzed fragments across all chromosomes. Although these particular Bis-PAGE conversion and quality control methods were exemplified in the context of a fragment library for SOLiD, the concepts can be generalized to include other genome-wide library constructions intended for DNA methylation analysis by alternative high-throughput or massively parallelized methods that are currently available.     

A comparison of existing global DNA methylation assays to low-coverage whole-genome bisulfite sequencing for epidemiological studies

DNA methylation is an epigenetic mark at the interface of genetic and environmental factors relevant to human disease. Quantitative assessments of global DNA methylation levels have therefore become important tools in epidemiology research, particularly for understanding effects of environmental exposures in complex diseases. Among the available methods of quantitative DNA methylation measurements, bisulfite sequencing is considered the gold standard, but whole-genome bisulfite sequencing (WGBS) has previously been considered too costly for epidemiology studies with high sample numbers. Pyrosequencing of repetitive sequences within bisulfite-treated DNA has been routinely used as a surrogate for global DNA methylation, but a comparison of pyrosequencing to WGBS for accuracy and reproducibility of methylation levels has not been performed. This study compared the global methylation levels measured from uniquely mappable (non-repetitive) WGBS sequences to pyrosequencing assays of several repeat sequences and repeat assay-matched WGBS data and determined uniquely mappable WGBS data to be the most reproducible and accurate measurement of global DNA methylation levels. We determined sources of variation in repetitive pyrosequencing assays to be PCR amplification bias, PCR primer selection bias in methylation levels of targeted sequences, and inherent variability in methylation levels of repeat sequences. Low-coverage, uniquely mappable WGBS showed the strongest correlation between replicates of all assays. By using multiplexing by indexed bar codes, the cost of WGBS can be lowered significantly to improve the accuracy of global DNA methylation assessments for human studies.