Differential methylation analysis for bisulfite sequencing using DSS

Bisulfite sequencing(BS-seq)technology measures DNA methylation at single nucleotide resolution.A key task in BSseq 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 in poultry:a review

As an important epigenetic modification,DNA methylation is involved in many biological processes such as animal cell differentiation,embryonic development,genomic imprinting and sex chromosome inactivation.As DNA methylation sequencing becomes more sophisticated,it becomes possible to use it to solve more zoological problems.This paper reviews the characteristics of DNA methylation,with emphasis on the research and application of DNA methylation in poultry.

Imprinting at the KBTBD6 locus involves species-specific m ternal methylation and monoallelic expression in livestock animals

Background The primary differentially methylated regions(DMRs) which are maternally hypermethylated serve as imprinting control regions(ICRs) that drive monoallelic gene expression, and these ICRs have been investigated due to their implications in mammalian development. Although a subset of genes has been identified as imprinted, in-depth comparative approach needs to be developed for identification of species-specific imprinted genes. Here, we examined DNA methylation status and allelic expression at the KBTBD6 locus across species and tissues and explored potential mechanisms of imprinting.Results Using whole-genome bisulfite sequencing and RNA-sequencing on parthenogenetic and normal porcine embryos, we identified a maternally hypermethylated DMR between the embryos at the KBTBD6 promoter Cp G island and paternal monoallelic expression of KBTBD6. Also, in analyzed domesticated mammals but not in humans, non-human primates and mice, the KBTBD6 promoter Cp G islands were methylated in oocytes and/or allelically methyl-ated in tissues, and monoallelic KBTBD6 expression was observed, indicating livestock-specific imprinting. Further analysis revealed that these Cp G islands were embedded within transcripts in porcine and bovine oocytes which coexisted with an active transcription mark and DNA methylation, implying the presence of transcription-dependent imprinting.Conclusions In this study, our comparative approach revealed an imprinted expression of the KBTBD6 gene in domesticated mammals, but not in humans, non-human primates, and mice which implicates species-specific evolution of genomic imprinting.

Reduced non-CpG methylation is a potential epigenetic target after spinal cord injury

DNA methylation is a critical epigenetic regulator in the occurrence and development of diseases and is closely related to various functional responses in relation to spinal cord injury.To investigate the role of DNA methylation in spinal cord injury,we constructed a library with reduced-representation bisulfite sequencing data obtained at various time points(day 0-42)after spinal cord injury in mice.Global DNA methylation levels,specifically non-CpG(CHG and CHH)methylation levels,decreased modestly following spinal cord injury.Stages post-spinal cord injury were classified as early(day 0-3),intermediate(day7-14),and late(day 28-42)based on similarity and hie rarchical cluste ring of global DNA methylation patterns.The non-CpG methylation level,which included CHG and CHH methylation levels,was markedly reduced despite accounting for a minor proportion of total methylation abundance.At multiple genomic sites,including the 5’untranslated regions,promoter,exon,intron,and 3’untranslated regions,the non-CpG methylation level was markedly decreased following spinal cord injury,whereas the CpG methylation level remained unchanged at these locations.Approximately one-half of the differentially methylated regions were located in intergenic areas;the other differentially methylated regions in both CpG and non-CpG regions were cluste red in intron regions,where the DNA methylation level was highest.The function of genes associated with differentially methylated regions in promoter regions was also investigated.From Gene Ontology analysis results,DNA methylation was implicated in a number of essential functional responses to spinal cord injury,including neuronal synaptic connection creation and axon regeneration.Notably,neither CpG methylation nor non-CpG methylation was implicated in the functional response of glial or inflammatory cells.In summary,our work elucidated the dynamic pattern of DNA methylation in the spinal co rd following injury and identified reduced nonCpG methylation as an epigenetic target after spinal cord injury in mice.

Non-invasive Prenatal Diagnosis of Trisomy 21 by Dosage Ratio of Fetal Chromosome-specific Epigenetic Markers in Maternal Plasma

This study examined the methylation difference in AIRE and RASSF1A between maternal and placental DNA, and the implication of this difference in the identification of free fetal DNA in maternal plasma and in prenatal diagnosis of trisomy 21. Maternal plasma samples were collected from 388 singleton pregnancies, and placental or chorionic villus tissues from 112 of them. Methylation-specific PCR （MSP） and methylation-sensitive restriction enzyme digestion followed by fluorescent quantitative PCR （MSRE ＋ PCR） were employed to detect the maternal-fetal methylation difference in AIRE and RASSF1A. Diagnosis of trisomy 21 was established according to the ratio of fetal-specific AIRE to RASSF1A in maternal plasma. Both methods confirmed that AIRE and RASSF1A were hypomethylated in maternal blood cells but hypermethylated in placental or chorionic villus tissues. Moreover, the differential methylation for each locus could be seen during the whole pregnant period. The positive rates of fetal AIRE and RASSF1A in maternal plasma were found to be 78.1% and 82.1% by MSP and 94.8% and 96.9% by MSRE ＋ PCR. MSRE ＋ PCR was superior to MSP in the identification of fetal-specific hypermethylated sequences （P〈0.05）. Based on the data from 266 euploidy pregnancies, the 95% reference interval of the fetal AIRE/RASSF1A ratio in maternal plasma was 0.33-1.77, which was taken as the reference value for determining the numbers of fetal chromosome 21 in 102 pregnancies. The accu-racy rate in 98 euploidy pregnancies was 96.9% （95/98）. Three of the four trisomy 21 pregnancies were confirmed with this method. It was concluded that hypermethylated AIRE and RASSF1A may serve as fetal-specific markers for the identification of fetal DNA in maternal plasma and may be used for noninvasive prenatal diagnosis of trisomy 21.

InfiniumPurify: An R package for estimating and accounting for tumor purity in cancer methylation research

The proposition of cancer cells in a tumor sample,named as tumor purity,is an intrinsic factor of tumor samples and has potentially great influence in variety of analyses including differential methylation,subclonal deconvolution and subtype clustering.Infinium-Purify is an integrated R package for estimating and accounting for tumor purity based on DNA methylation Infinium 450 k array data.InfiniumPurify has three main functions getPurity,InfiniumDMC and InfiniumClust,which could infer tumor purity,differential methylation analysis and tumor sample cluster accounting for estimated or user-provided tumor purities,respectively.The InfiniumPurify package provides a comprehensive analysis of tumor purity in cancer methylation research.

The progress on the estimation of DNA methylation level and the detection of abnormal methylation

Background:DNA methylation is a key heritable epigenetic modification that plays a crucial role in transcriptional regulation and therefore a broad range of biological processes.The complex patterns of DNA methylation highlight the significance of the profiling the DNA methylation landscape.Results:In this review,the main high-throughput detection technologies are summarized,and then the three trends of computational estimation of DNA methylation levels were analyzed,especially the expanding of the methylation data with lower coverage.Furthermore,the detection methods of differential methylation patterns for sequencing and array data were presented.Conclusions:More and more research indicated the great importance of DNA methylation changes across different diseases,such as cancers.Although a lot of enormous progress has been made in understanding the role of DNA methylation,only few methylated genes or functional elements serve as clinically relevant cancer biomarkers.The bottleneck in DNA methylation advances has shifted from data generation to data analysis.Therefore,it is meaningful to develop machine learning models for computational estimation of methylation profiling and identify the potential biomarkers.

Genome-Wide Analysis of DNA Methylation in Soybean

Cytosine methylation is an important mechanism for dynamical regulation of gene expression and trans- posable element （TE） mobility during plant developmental processes. Here, we identified the transcription start sites of genes using high-throughput sequencing and then analyzed the DNA methylation status in soybean roots, stems, leaves, and cotyledons of developing seeds at single-base resolution. Profiling of DNA methylation in different organs revealed 2162 differentially methylated regions among organs, and a portion of hypomethylated regions were correlated with high expression of neighboring genes. Because of the different distribution of class I TEs （retrotransposons） and class II TEs （DNA transposons）, the promoters of the lowest-expressed genes showed higher levels of CG and CHG methyla- tion but a lower level of CHH methylation. We further found that the CHH methylation level of class II TEs was higher than class I TEs, possibly due to the presence of more smRNAs in class II TEs. In cotyledons of developing seeds, smRNA abundance was roughly positively correlated with hypermethylated regions but negatively related to hypomethylated regions. These studies provide significant insights into the complicated interplays among DNA methylation, smRNA abundance, TE distribution, and gene expression in soybean.

Epigenome-wide DNA methylation study of whole blood in patients with sporadic amyotrophic lateral sclerosis

Background:Epigenetics,and especially DNA methylation,contributes to the pathogenesis of sporadic amyotrophic lateral sclerosis(SALS).This study aimed to investigate the role of DNA methylation in SALS using whole blood of SALS patients.Methods::In total,32 SALS patients and 32 healthy controls were enrolled in this study.DNA was isolated from whole blood collected from the participants.DNA methylation profiles were generated using Infinium MethylationEPIC BeadChip.Results:We identified 34 significant differentially methylated positions(DMPs)in whole blood from SALS patients,compared with the healthy controls.Of these DMPs,five were hypermethylated and 29 were hypomethylated;they corresponded to 13 genes.For the DMPs,ATAD3B and BLK were hypermethylated,whereas DDO,IQCE,ABCB1,DNAH9,FIGN,NRP1,TMEM87B,CCSAP,ST6GALNAC5,MYOM2,and RUSC1-AS1 were hypomethylated.We also identified 12 differentially methylated regions(DMRs),related to 12 genes(NWD1,LDHD,CIS,IQCE,TNF,PDE1C,LGALS1,CSNK1E,LRRC23,ENO2,ELOVL2,and ELOVL2-AS1).According to data from the Kyoto Encyclopedia of Genes and Genomes database,DNAH9 and TNF are involved in the amyotrophic lateral sclerosis(ALS)pathway.Correlation analysis between clinical features and DNA methylation profiling indicated that the methylation level of ELOVL2 and ARID1B was positively associated with the age of onset(r=0.86,adjust P=0.001)and disease duration(r=0.83,adjust P=0.01),respectively.Conclusions:We found aberrant methylation in DMP-and DMR-related genes,implying that many epigenetic alterations,such as the hypomethylation of DNAH9 and TNF,play important roles in ALS etiology.These findings can be helpful for developing new therapeutic interventions.

Normal epigenetic inheritance in mice conceived by in vitro fertilization and embryo transfer

An association between assisted reproductive technology （ART） and neurobehavioral imprinting disorders has been reported in many studies, and it seems that ART may interfere with imprint reprogramming. However, it has never been explored whether epigenetic erros or imprinting disease susceptibility induced by ART can be inherited transgenerationally. Hence, the aim of this study was to determine the effect of in vitro fertilization and embryo transfer （IVF-ET） on transgenerational inheritance in am inbred mouse model. Mice derived from IVF-ET were outcrossed to wild-type C57BL/6J to obtain their female and male line F2 and F3 generations. Their behavior, morphology, histology, and DNA methylation status at several important differentially methylated regions （DMRs） were analyzed by Morris water maze, hematoxylin and eosin （H＆E） staining, and bisulfite genomic sequencing. No significant differences in spatial learning or phenotypic abnormality were found in adults derived from IVF （F1） and female and male line F2 and F3 generations. A borderline trend of hypomethylation was found in H19 DMR CpG island 3 in the female line-derived F3 generation （0.40±0.118, P=0.086）. Methylation status in H19/Igf2 DMR island 1, Igf2 DMR, KvDMR, and Snrpn DMR displayed normal patterns. Methylation percentage did not differ significantly from that of adults conceived naturally, and the expression of the genes they regulated was not disturbed. Transgenerational integrity, such as behavior, morphology, histology, and DNA methylation status, was maintained in these generations, which indicates that exposure of female germ cells to hormonel stimulation and gamete manipulation might not affect the individuals and their descendents.

Functional spermatid-like cells derived from the ground-state embryonic stem cells in vitro

Embryonic stem cells（ESCs）are pluripotent and can self-assemble to form cell clusters or embryoid bodies（EBs）,which can then differentiate into all cell types of the three germ layers,as among which one is the primordial germ cell（PGCs）（Daley,2007）.In vivo PGCs are the gamete founder cells（the ooctyes and sperms）,which transmit genetic information from one generation to the next generation to maintain mammalian life cycles.About 15%of couples in China are infertile;