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.     

DNA methylation: an identity card for brain cells

A recently published study has revealed the genome-wide dynamics of DNA methylation and hydroxymethylation patterns at single-base resolution in the human and mouse developing brain.     

Age-related epigenome-wide DNA methylation and hydroxymethylation in longitudinal mouse blood

DNA methylation at cytosine-phosphate-guanine (CpG) dinucleotides changes as a function of age in humans and animal models, a process that may contribute to chronic disease development. Recent studies have investigated the role of an oxidized form of DNA methylation – 5-hydroxymethylcytosine (5hmC) – in the epigenome, but its contribution to age-related DNA methylation remains unclear. We tested the hypothesis that 5hmC changes with age, but in a direction opposite to 5-methylcytosine (5mC), potentially playing a distinct role in aging. To characterize epigenetic aging, genome-wide 5mC and 5hmC were measured in longitudinal blood samples (2, 4, and 10 months of age) from isogenic mice using two sequencing methods – enhanced reduced representation bisulfite sequencing and hydroxymethylated DNA immunoprecipitation sequencing. Examining the epigenome by age, we identified 28,196 unique differentially methylated CpGs (DMCs) and 8,613 differentially hydroxymethylated regions (DHMRs). Mouse blood showed a general pattern of epigenome-wide hypermethylation and hypo-hydroxymethylation with age. Comparing age-related DMCs and DHMRs, 1,854 annotated genes showed both differential 5mC and 5hmC, including one gene – Nfic – at five CpGs in the same 250 bp chromosomal region. At this region, 5mC and 5hmC levels both decreased with age. Reflecting these age-related epigenetic changes, Nfic RNA expression in blood decreased with age, suggesting that age-related regulation of this gene may be driven by 5hmC, not canonical DNA methylation. Combined, our genome-wide results show age-related differential 5mC and 5hmC, as well as some evidence that changes in 5hmC may drive age-related DNA methylation and gene expression.     

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.     

Comparative genome-wide DNA methylation analysis of colorectal tumor and matched normal tissues

Aberrant DNA methylation often occurs in colorectal cancer (CRC). In our study we applied a genome-wide DNA methylation analysis approach, MethylCap-seq, to map the differentially methylated regions (DMRs) in 24 tumors and matched normal colon samples. In total, 2687 frequently hypermethylated and 468 frequently hypomethylated regions were identified, which include potential biomarkers for CRC diagnosis. Hypermethylation in the tumor samples was enriched at CpG islands and gene promoters, while hypomethylation was distributed throughout the genome. Using epigenetic data from human embryonic stem cells, we show that frequently hypermethylated regions coincide with bivalent loci in human embryonic stem cells. DNA methylation is commonly thought to lead to gene silencing; however, integration of publically available gene expression data indicates that 75% of the frequently hypermethylated genes were most likely already lowly or not expressed in normal tissue. Collectively, our study provides genome-wide DNA methylation maps of CRC, comprehensive lists of DMRs, and gives insights into the role of aberrant DNA methylation in CRC formation.     

Comparative genome-wide DNA methylation analysis of colorectal tumor and matched normal tissues

Aberrant DNA methylation often occurs in colorectal cancer (CRC). In our study we applied a genome-wide DNA methylation analysis approach, MethylCap-seq, to map the differentially methylated regions (DMRs) in 24 tumors and matched normal colon samples. In total, 2687 frequently hypermethylated and 468 frequently hypomethylated regions were identified, which include potential biomarkers for CRC diagnosis. Hypermethylation in the tumor samples was enriched at CpG islands and gene promoters, while hypomethylation was distributed throughout the genome. Using epigenetic data from human embryonic stem cells, we show that frequently hypermethylated regions coincide with bivalent loci in human embryonic stem cells. DNA methylation is commonly thought to lead to gene silencing; however, integration of publically available gene expression data indicates that 75% of the frequently hypermethylated genes were most likely already lowly or not expressed in normal tissue. Collectively, our study provides genome-wide DNA methylation maps of CRC, comprehensive lists of DMRs, and gives insights into the role of aberrant DNA methylation in CRC formation.     

Nightshift work,chronotype, and genome-wide DNA methylation in blood

Molecular mechanisms underlying the negative health effects of shift work are poorly understood, which remains a barrier to developing intervention strategies to protect the long-term health of shift workers. We evaluated genome-wide differences in DNA methylation (measured in blood) between 111 actively employed female nightshift and 86 actively employed female dayshift workers from the Seattle metropolitan area. We also explored the effect of chronotype (i.e., measure of preference for activity earlier or later in the day) on DNA methylation among 110 of the female nightshift workers and an additional group of 131 male nightshift workers. Methylation data were generated using the Illumina Infinium HumanMethylation450 BeadChip (450K) Array. After applying the latest methylation data processing methods, we compared methylation levels at 361,210 CpG loci between the groups using linear regression models adjusted for potential confounders and applied the false-discovery rate (FDR) ≤ 0.05 to account for multiple comparisons. No statistically significant associations at the genome-wide level were observed with shift work or chronotype, though based on raw P values and absolute effect sizes, there were suggestive associations in genes that have been previously linked with cancer (e.g., BACH2, JRK, RPS6KA2) and type-2 diabetes (e.g., KCNQ1). Given that our study was underpowered to detect moderate effects, examining these suggestive results in well-powered independent studies or in pooled data sets may improve our understanding of the pathways underlying the negative health effects of shift work and the influence of personal factors such as chronotype. Such an approach may help identify potential interventions that can be used to protect the long-term health of shift workers.     

Comparison of bisulfite sequencing PCR with pyrosequencing for measuring differences in DNA methylation

DNA methylation strongly affects chromatin structure and the regulation of gene expression. For many years, bisulfite sequencing PCR (BSP) has served as the “gold standard” for measuring DNA methylation. However, with the evolution of pyrosequencing as a tool to evaluate DNA methylation, the need arises to compare the relative efficiencies of the two techniques in measuring DNA methylation. We provide for the first time a direct assessment of BSP and pyrosequencing to detect and quantify hypomethylation, hypermethylation, and mixed methylation of the ABCB1 promoter in various drug-sensitive and drug-resistant MCF-7 breast cancer cell lines through head-to-head experimentation. Our findings indicate that although both methods can reliably detect increased, decreased, and mixed methylation of DNA, BSP appears to be more sensitive than pyrosequencing at detecting strong hypermethylation of DNA. However, we also observed greater variability in the methylation of CpG sites by BSP, possibly due to the additional bacterial cloning step required by BSP over pyrosequencing. BSP and pyrosequencing equally detected hypomethylation and mixed methylation of DNA. The ability of pyrosequencing to reliably detect differences in DNA methylation across cell populations without requiring the cloning of bisulfite-treated DNA into bacterial expression vectors was seen as a major advantage of this technique.     

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.     

Cas9-targeted Nanopore sequencing rapidly elucidates the transposition preferences and DNA methylation profiles of mobile elements in plants

Transposable element insertions (TEIs) are an important source of genomic innovation by contributing to plant adaptation, speciation, and the production of new varieties. The often large, complex plant genomes make identifying TEIs from short reads difficult and expensive. Moreover, rare somatic insertions that reflect mobilome dynamics are difficult to track using short reads. To address these challenges, we combined Cas9-targeted Nanopore sequencing (CANS) with the novel pipeline NanoCasTE to trace both genetically inherited and somatic TEIs in plants. We performed CANS of the EVADÉ (EVD) retrotransposon in wild-type Arabidopsis thaliana and rapidly obtained up to 40× sequence coverage. Analysis of hemizygous T-DNA insertion sites and genetically inherited insertions of the EVD transposon in the ddm1 (decrease in DNA methylation 1) genome uncovered the crucial role of DNA methylation in shaping EVD insertion preference. We also investigated somatic transposition events of the ONSEN transposon family, finding that genes that are downregulated during heat stress are preferentially targeted by ONSENs. Finally, we detected hypomethylation of novel somatic insertions for two ONSENs. CANS and NanoCasTE are effective tools for detecting TEIs and exploring mobilome organization in plants in response to stress and in different genetic backgrounds, as well as screening T-DNA insertion mutants and transgenic plants.     

Temperature-independent genome-wide DNA methylation profile in turbot post-embryonic development

The morphological and biological characteristics of ectothermic vertebrates are known to be strongly influenced by environmental conditions, particularly temperature. Epigenetic mechanisms such as DNA methylation have been reported to contribute to the phenotypic plasticity observed in vertebrates in response to environmental changes. Additionally, DNA methylation is a dynamic process that occurs throughout vertebrate ontogeny and it has been associated with the activation and silencing of gene expression during post-embryonic development and metamorphosis. In this study, we investigated genome-wide DNA methylation profiles during turbot metamorphosis, as well as the epigenetic effects of temperature on turbot post-embryonic development. Fish growth and rates of development were greatly affected by rearing temperature. Thus, turbot raised at ambient temperature (18 °C) achieved greater body weights and progressed through development more quickly than those reared at a colder temperature (14 °C). Genome-wide DNA methylation dynamics analyzed via a methylation-sensitive amplified polymorphism (MSAP) technique were not significantly different between animals reared within the two different thermal environments. Furthermore, comparisons between phenotypically similar fish revealed that genome-wide DNA methylation profiles do not necessarily correlate with specific developmental stages in turbot.     

Reprogramming DNA methylation in the mammalian life cycle: building and breaking epigenetic barriers

In mammalian development, epigenetic modifications, including DNA methylation patterns, play a crucial role in defining cell fate but also represent epigenetic barriers that restrict developmental potential. At two points in the life cycle, DNA methylation marks are reprogrammed on a global scale, concomitant with restoration of developmental potency. DNA methylation patterns are subsequently re-established with the commitment towards a distinct cell fate. This reprogramming of DNA methylation takes place firstly on fertilization in the zygote, and secondly in primordial germ cells (PGCs), which are the direct progenitors of sperm or oocyte. In each reprogramming window, a unique set of mechanisms regulates DNA methylation erasure and re-establishment. Recent advances have uncovered roles for the TET3 hydroxylase and passive demethylation, together with base excision repair (BER) and the elongator complex, in methylation erasure from the zygote. Deamination by AID, BER and passive demethylation have been implicated in reprogramming in PGCs, but the process in its entirety is still poorly understood. In this review, we discuss the dynamics of DNA methylation reprogramming in PGCs and the zygote, the mechanisms involved and the biological significance of these events. Advances in our understanding of such natural epigenetic reprogramming are beginning to aid enhancement of experimental reprogramming in which the role of potential mechanisms can be investigated in vitro. Conversely, insights into in vitro reprogramming techniques may aid our understanding of epigenetic reprogramming in the germline and supply important clues in reprogramming for therapies in regenerative medicine.     

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.     

Isolation and characterization of polymorphic microsatellite loci in Indian major carp,Catla catla using next-generation sequencing platform

Catla catla, the second most important Indian major carp, is gaining its popularity among Indian fish farmers due to its high growth rate and consumer preferences. Simple sequence repeats (SSRs) are rapidly evolving, versatile, co-dominant and highly informative molecular markers used in genetic research. However, the time and cost involved in developing such resources has limited their extensive use. Advent of massive parallel sequencing technology has considerably eased these limitations. In the present investigation, we used Ion Torrent sequencing platform to identify potentially amplifiable microsatellite loci for catla. A modest sequencing volume generated approximately 5.7 MB of sequence data. Out of 29,794 sequences generated, 21,477 contained simple sequence repeats. Only 81 sequences had enough flanking sequences for primer designing. Out of 81 loci, 51 were successfully PCR amplified in a panel of five unrelated individuals. Out of 15 loci randomly checked for polymorphism, 13 loci were polymorphic with allele number ranged from 3 to 6 and two loci were found to be monomorphic. The observed and expected heterozygosities ranged from 0.565 to 0.870 and 0.483–0.804, respectively. These markers will be useful for studying genetics of wild populations, breeding programs of C. catla and closely related species.     

Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

Background

Influenza viruses exist as a large group of closely related viral genomes, also called quasispecies. The composition of this influenza viral quasispecies can be determined by an accurate and sensitive sequencing technique and data analysis pipeline. We compared the suitability of two benchtop next-generation sequencers for whole genome influenza A quasispecies analysis: the Illumina MiSeq sequencing-by-synthesis and the Ion Torrent PGM semiconductor sequencing technique.

Results

We first compared the accuracy and sensitivity of both sequencers using plasmid DNA and different ratios of wild type and mutant plasmid. Illumina MiSeq sequencing reads were one and a half times more accurate than those of the Ion Torrent PGM. The majority of sequencing errors were substitutions on the Illumina MiSeq and insertions and deletions, mostly in homopolymer regions, on the Ion Torrent PGM. To evaluate the suitability of the two techniques for determining the genome diversity of influenza A virus, we generated plasmid-derived PR8 virus and grew this virus in vitro. We also optimized an RT-PCR protocol to obtain uniform coverage of all eight genomic RNA segments. The sequencing reads obtained with both sequencers could successfully be assembled de novo into the segmented influenza virus genome. After mapping of the reads to the reference genome, we found that the detection limit for reliable recognition of variants in the viral genome required a frequency of 0.5% or higher. This threshold exceeds the background error rate resulting from the RT-PCR reaction and the sequencing method. Most of the variants in the PR8 virus genome were present in hemagglutinin, and these mutations were detected by both sequencers.

Conclusions

Our approach underlines the power and limitations of two commonly used next-generation sequencers for the analysis of influenza virus gene diversity. We conclude that the Illumina MiSeq platform is better suited for detecting variant sequences whereas the Ion Torrent PGM platform has a shorter turnaround time. The data analysis pipeline that we propose here will also help to standardize variant calling in small RNA genomes based on next-generation sequencing data.     

Improved reproducibility in genome-wide DNA methylation analysis for PAXgene-fixed samples compared with restored formalin-fixed and paraffin-embedded DNA

Formalin fixation has been the standard method for conservation of clinical specimens for decades. However, a major drawback is the high degradation of nucleic acids, which complicates its use in genome-wide analyses. Unbiased identification of biomarkers, however, requires genome-wide studies, precluding the use of the valuable archives of specimens with long-term follow-up data. Therefore, restoration protocols for DNA from formalin-fixed and paraffin-embedded (FFPE) samples have been developed, although they are cost-intensive and time-consuming. An alternative to FFPE and snap-freezing is the PAXgene Tissue System, developed for simultaneous preservation of morphology, proteins, and nucleic acids. In the current study, we compared the performance of DNA from either PAXgene or formalin-fixed tissues to snap-frozen material for genome-wide DNA methylation analysis using the Illumina 450K BeadChip. Quantitative DNA methylation analysis demonstrated that the methylation profile in PAXgene-fixed tissues showed, in comparison with restored FFPE samples, a higher concordance with the profile detected in frozen samples. We demonstrate, for the first time, that DNA from PAXgene conserved tissue performs better compared with restored FFPE DNA in genome-wide DNA methylation analysis. In addition, DNA from PAXgene tissue can be directly used on the array without prior restoration, rendering the analytical process significantly more time- and cost-effective.     

DNA sequencing research group: 2006 general survey of DNA sequencing facilities.

Over the past few years, technological advances in automated DNA sequencing have had a profound effect on the nature of DNA sequencing laboratories. To characterize the changes occurring within DNA sequencing facilities, the DNA Sequencing Research Group conducted three previous studies, in 1998, 2000, and 2003. A new general survey has been designed and conducted by the DSRG to capture the current status of DNA sequencing facilities in all sectors. Included were questions regarding facility administration, pricing, instrumentation, technology, protocols, and operation. The results of the survey are presented here, accompanied by comparisons to the previous surveys. These comparisons formed a basis for the discussion of trends within the facilities in response to the dynamics of a changing technology.