Characterization of epigenetic alterations in esophageal cancer by whole-genome bisulfite sequencing

Esophageal carcinoma is a common and aggressive malignancy, and its patients have dismal clinical outcomes. The epigenetic dysregulation in both major subtypes, esophageal squamous cell carcinoma (ESCC) and adenocarcinoma (EAC), awaits further characterization. Here, we perform whole-genome bisulfite sequencing (WGBS) on a total of 43 esophageal cancer and normal samples, generating one of the largest WGBS datasets in this cancer to date. Focusing on hypomethylated regions in cancer, we show that they are associated with increased chromatin activity and enhancer RNA expression. Using this large collection of WGBS dataset, we reveal and validate novel clusters in both ESCC and EAC. We further identify specific molecular features in each cluster, with potential clinical implications. These data together advance our understanding of the epigenetic alterations in esophageal cancer and provide a rich resource for the research community of this disease.

164 Genome-wide DNA Methylation Profiling in in Utero Heat-stressed Pigs as Measured by Whole-genome Bisulfite Sequencing

In utero heat stress (IUHS) has several postnatal consequences in pigs that compromise health, increase stress response, and reduce performance. These phenotypes may be caused by epigenetic modifications such as DNA methylation, which are heritable molecular modifications that impact gene expression and phenotypic outcomes without changing the DNA sequence. Therefore, we aimed to compare the DNA methylation profiles between in-utero thermoneutral (IUTN) and IUHS pigs to identify differentially methylated regions. Twenty-four pregnant gilts were evenly assigned to either a thermoneutral (17.5 ± 2.1°C) or heat stress (cycling 26 to 36°C) chamber from d 0 to 59 of gestation, followed by thermoneutral conditions (20.9 ± 2.3°C) for the rest of gestation and until the piglets were weaned. At 105 d of age, 10 IUTN and 10 IUHS piglets were euthanized and Longissimus dorsi muscle samples were collected and used to perform whole-genome bisulfite sequencing (WGBS). Purified genomic DNA was fragmented and bisulfite conversion was performed. Illumina platforms were used to sequence WGBS libraries. All pigs had similar proportions of methylation at CpG sites. Two-hundred-sixty-eight genomic regions were differentially methylated between IUTN and IUHS pigs. These identified regions are located across all pig chromosomes and ranged from 2 (SSC18) to 40 (SSC10). Eighty-five unique differentially-methylated genes were identified. These genes have been reported to be involved in key biological processes such as transcriptional repressor activity and tRNA processing (e.g., SKOR2,TRMT6, TSEN2), cellular response to heat stress (e.g.,CCAR2), placental vascularization (e.g.,FZD5), central nervous system (e.g.,VEPH1), cholesterol biosynthesis (e.g., CYB5R1), insulin receptor substrate (e.g.,IRS2), synaptic transmission (e.g.,RIMBP2), neurotrophic factor receptor activity (e.g.,LIFR), immune response (e.g., CD84), DNA repair (e.g., CHD1L), and cell proliferation and endocrine signaling (e.g., SSTR1, CYB5R1). These findings contribute to a better understanding of the epigenomic mechanisms underlying postnatal consequences of IUHS in pigs.

OMIC-06. MOLECULAR SUBGROUPING OF MEDULLOBLASTOMA VIA LOW-DEPTH WHOLE GENOME BISULFITE SEQUENCING

Introduction International consensus recognises four molecular subgroups of medulloblastoma, each with distinct molecular features and clinical outcomes. The current gold-standard for subgroup assignment is DNA methylation microarray. There is an unmet need to develop platform-independent subgrouping assays which are both non-proprietary and compatible with rapidly-expanding WGS capacity in healthcare. Whole Genome Bisulfite Sequencing (WGBS) enables the assessment of genome-wide methylation status at single-base resolution. Previously, WGBS adoption has been limited by cost and sample quality/quantity requirements. Its application for routine detection of medulloblastoma subgroups has not previously been reported. Methodology Two datasets were utilised; 36 newly-sequenced low-depth (10x coverage) and 34 publicly-available high-depth (30x) WGBS medulloblastomas, all with matched DNA methylation microarray data. We compared platform concordance and identified molecular subgroups. Machine-learning WGBS-based subgroup classifiers were optimised and compared between platforms. Aneuploidy and mutation detection using WGBS was optimised and compared to microarray-derived estimates where possible. Finally, comprehensive subgroup-specific DNA methylation signatures were identified. Results We optimised a pipeline for processing, quality control and analysis of low-depth WGBS data, suitable for routine molecular subgrouping and aneuploidy assessment. We demonstrated the suitability of fresh-frozen and FFPE DNA for WGBS, and, using downsampling, showed that subgroup calling is robust at coverages as low as 2x. We identified differentially methylated regions that, due to poor representation, could not be detected using methylation microarrays. Molecular subgroups of medulloblastoma assigned using WGBS were concordant with array-based definitions, and WGBS-derived classifier performance measures exceeded microarray-derived classifiers. Conclusion We describe a platform-independent assay for molecular subgrouping of medulloblastoma using WGBS. It performs equivalently to current array-based methods at comparable cost ($405 vs $596) and provides a proof-of-concept for its routine clinical adoption using standard WGS technology. Finally, the full methylome enabled elucidation of additional biological heterogeneity that has hitherto been inaccessible.

DNA methylation affects pre-mRNA transcriptional initiation and processing in Arabidopsis

Background: DNA methylation may regulate pre-mRNA transcriptional initiation and processing, thus affecting gene expression. Unlike animal cells, plants, especially Arabidopsis thaliana, have relatively low DNA methylation levels, limiting our ability to observe any correlation between DNA methylation and pre-mRNA processing using typical short-read sequencing. However, with newly developed long-read sequencing technologies, such as Oxford Nanopore Technology Direct RNA sequencing (ONT DRS), combined with whole-genome bisulfite sequencing, we were able to precisely analyze the relationship between DNA methylation and pre-mRNA transcriptional initiation and processing using DNA methylation-related mutants. Results: Using ONT DRS, we generated more than 2 million high-quality full-length long reads of native mRNA for each of the wild type Col-0 and mutants defective in DNA methylation, identifying a total of 117,474 isoforms. We found that low DNA methylation levels around splicing sites tended to prevent splicing events from occurring. The lengths of the poly(A) tail of mRNAs were positively correlated with DNA methylation. DNA methylation before transcription start sites or around transcription termination sites tended to result in gene-silencing or read-through events. Furthermore, using ONT DRS, we identified novel transcripts that we could not have otherwise, since transcripts with intron retention and fusion transcripts containing the uncut intergenic sequence tend not to be exported to the cytoplasm. Using the met1-3 mutant with activated constitutive heterochromatin regions, we confirmed the effects of DNA methylation on pre-mRNA processing. Conclusion: The combination of ONT DRS with whole-genome bisulfite sequencing was a powerful tool for studying the effects of DNA methylation on splicing site selection and pre-mRNA processing, and therefore regulation of gene expression.

Ultra-deep whole genome bisulfite sequencing reveals a single methylation hotspot in human brain mitochondrial DNA

ABSTRACTWhile DNA methylation is established as a major regulator of gene expression in the nucleus, the existence of mitochondrial DNA (mtDNA) methylation remains controversial. Here, we characterised the mtDNA methylation landscape in the prefrontal cortex of neurological healthy individuals (n=26) and patients with Parkinson’s disease (n=27), using a combination of whole genome bisulfite sequencing (WGBS) and bisulfite-independent methods. Accurate mtDNA mapping from WGBS data required alignment to an mtDNA reference only, to avoid misalignment to nuclear mitochondrial pseudogenes. Once correctly aligned, WGBS data provided ultra-deep mtDNA coverage (16,723±7,711), and revealed overall very low levels of cytosine methylation. The highest methylation levels (5.49±0.97%) were found on CpG position m.545, located in the heavy-strand promoter 1 region. The m.545 methylation was validated using a combination of methylation-sensitive DNA digestion and quantitative PCR analysis. We detected no association between mtDNA methylation profile and Parkinson’s disease. Interestingly, m.545 methylation correlated with the levels of mtDNA transcripts, suggesting a putative role in regulating mtDNA gene expression. In addition, we propose a robust framework for methylation analysis of mtDNA from WGBS data, which is less prone to false-positive findings due to misalignment of nuclear mitochondrial pseudogene sequences.Graphical abstract of the analyses and main findingsFresh-frozen brain tissue was obtained from the prefrontal cortex (Brodmann area 9) of 53 individuals, comprising 27 patients with idiopathic PD and 26 healthy controls. Tissue from the same samples was used in three different downstream analyses. WGBS was conducted on all 53 samples and the data were analysed using three different alignment strategies. Alignment against an mtDNA reference only was clearly superior as it gave the highest and most even depth of coverage. WGBS analysis revealed that mtDNA harbours very low levels of cytosine methylation, with the exception of the CpG position m.545 within the HSP1 region (lower right inset). The m.545 methylation was confirmed by bisulfite- and sequencing-independent methods, employing methylation-specific MspJI DNA digestion, followed by quantification with qPCR or fluorescent PCR and capillary electrophoresis. Finally, mtDNA transcript levels were determined by RT-qPCR and correlated to m.545 methylation levels, showing a positive association.

Integration of multi-omics approaches for functional characterization of muscle related selective sweep genes in Nanchukmacdon

Pig as a food source serves daily dietary demand to a wide population around the world. Preference of meat depends on various factors with muscle play the central role. In this regards, selective breeding abled us to develop “Nanchukmacdon” a pig breeds with an enhanced variety of meat and high fertility rate. To identify genomic regions under selection we performed whole-genome resequencing, transcriptome, and whole-genome bisulfite sequencing from Nanchukmacdon muscles samples and used published data for three other breeds such as Landrace, Duroc, Jeju native pig and analyzed the functional characterization of candidate genes. In this study, we present a comprehensive approach to identify candidate genes by using multi-omics approaches. We performed two different methods XP-EHH, XP-CLR to identify traces of artificial selection for traits of economic importance. Moreover, RNAseq analysis was done to identify differentially expressed genes in the crossed breed population. Several genes (UGT8, ZGRF1, NDUFA10, EBF3, ELN, UBE2L6, NCALD, MELK, SERP2, GDPD5, and FHL2) were identified as selective sweep and differentially expressed in muscles related pathways. Furthermore, nucleotide diversity analysis revealed low genetic diversity in Nanchukmacdon for identified genes in comparison to related breeds and whole-genome bisulfite sequencing data shows the critical role of DNA methylation pattern in identified genes that leads to enhanced variety of meat. This work demonstrates a way to identify the molecular signature and lays a foundation for future genomic enabled pig breeding.