{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Céline Vandecasteele"],"organism":["Coturnix japonica"],"software":["Longshot v0.4.1","megalodon v2.5.0","nf-core/methylseq v1.5"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-16868"],"description":["Whole Genome Bisulfite Sequencing (WGBS) has been the gold standard DNA methylation mapping and quantification for over a decade. Oxford Nanopore Technologies (ONT) sequencing directly measures nucleotide modifications. In this study, we have compared DNA methylation levels (5-methylcytosine) at CpG sites in the quail genome using WGBS and ONT. Samples were collected to investigate transgenerational DNA methylation changes in Japanese quail following ancestral exposure to a phytoestrogen. Blood samples from 24 third-generation (G3) individuals—descendants of either treated or untreated ancestors—were sequenced after bisulfite conversion. Both methods revealed broadly consistent methylation patterns. ONT reads covered more CpG sites and detected a higher number of differentially methylated cytosines (DMCs). Principal component analyses showed that both sex and ancestral treatment groups accounted for a portion of the observed epigenetic variation, for both technologies. Strong concordance between WGBS and ONT results supports the reliability of ONT sequencing for epigenomic research, including in quails. These data pave the way for further investigation into whether genistein induces epigenetic changes for several generations."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Nucleic Acid Extraction - High molecular weight genomic DNA (HMW DNA) was extracted from the quail blood samples using a high-salt extraction method (Roussot et al. 2003)","Sample Collection - Quails of the S+ line4 were bred at the PEAT Poultry Experimental Facility (INRAE, Nouzilly). Initially, 10 parent pairs were used and the fertilised eggs from each pair were randomly assigned to two groups (\\\"epilines\\\"): a control group (epi-) and a treated group (epi+) that received 500 μg genistein (natural endocrine disruptor, Sigma-Aldrich) prior to incubation. Genistein was first dissolved in 100% ethanol and then diluted 10-fold in sesame oil before injection. Genistein was injected into the yolk of freshly laid eggs on the first day of incubation. Two generations were then bred for each line using mirrored one-pair matings to minimise genetic variation between both epilines. This approach was designed to increase the likelihood that any difference observed in the third generation epilines was due to transgenerational inheritance of epigenetic modifications. According to this protocol, blood samples were taken from 24 third generation (G3) quails, 12 of which were controls and 12 of which were descending from treated ancestors (see Experimental design, archive.softwareheritage.org/swh:1:dir:6f62a8bc6f2d392cb91298ab11a05302a3d3e099). All quails were raised together, in the same floor pen, until blood sampling at 35 days of age. The experiments were carried out in accordance with European Union guidelines for the care of animals according to Council Directives 98/58/EC and 86/609/EEC. The facility was registered with the French Ministry of Agriculture under licence number C37-175-1 for animal experimentation. The experiments were approved by the local ethics committee for animal experimentation (Val de Loire) and by the French Ministry of Agriculture (authorization n°37-002). All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).","Library Construction - WGBS libraries were prepared according to the manufacturer's instructions (Biooscientific NEXTflex™ Bisulfite Library Prep Kit for Illumina Sequencing). Briefly, 1500 ng of DNA was sonicated, followed by size selection using AMPure XP (Beckman Coulter) beads and subsequent ligation of adaptors prior to sequencing. Bisulfite treatment was then performed using the EZ DNA Methylation-Gold™ Kit (Zymo Research) for 2.5 hours, followed by 12 cycles of PCR. Library quality was assessed using an Advanced Analytical Fragment Analyser (Agilent), while library quantification was performed by qPCR using the Kapa Library Quantification Kit.","Library Construction - ONT libraries were prepared according to the manufacturer's instructions (1D gDNA selecting for long reads, SQK-LSK109). DNA quantification was performed using the Qubit dsDNA HS Assay Kit (Invitrogen), DNA purity was assessed using a NanoDrop (Thermo Fisher Scientific) and size distribution was evaluated using Fragment Analyzer (Agilent). Purification was performed using AMPure XP beads (Beckman Coulter). For each flow cell, 5 µg of purified DNA was sheared at 25 kb using the Megaruptor system (Diagenode) and size selection was performed using the Short Read Eliminator M kit (Circulomics). DNA repair, end repair and adapter ligation steps were performed on 2 µg of DNA.","Sequencing - Sequencing of the WGBS libraries (2x150 bp) was performed on a NovaSeq 6000 (Illumina).","Sequencing - Libraries were loaded onto FLO-PRO002 R9.4.1 flow cells (PromethION instrument) at 20 fmol within 72h. Nuclease washes were performed at 24h and 48h."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - Longshot v0.4.1 was used to detect native SNPs directly present in the ONT sample alignments. The retrieved SNP list was merged between the 23 samples using bcftools v1.14. The combined VCF file was used to filter putative SNP positions (n=10,397,021) from the Bismark and Megalodon outputs.","Data Transformation - WGBS reads were cleaned using fastp v0.21 to remove artifactual polyG sequences at the end of the reads and to trim adapters for read1 and read2 respectively. After this cleaning step, the WGBS (paired-end) reads were processed using the nf-core/methylseq v1.5 tool. Briefly, reads were trimmed using Trim Galore v0.6.4 to remove adapters and low-quality bases, and trimmed reads were mapped to the quail reference genome using Bismark v0.22.3. Bismark extracts methylation sites and generates a cytosine report for each sample (--cytosine_report), considering all cytosines on both strands and reporting their position, strand, trinucleotide context and methylation state.","Data Transformation - CpG methylation from ONT reads were called with megalodon v2.5.0 along with guppy v5.0.17, using the res_dna_r941_prom _modbases_5mC_CpG_v001.cfg model with default parameters."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq 6000","PromethION"],"study_type":["methylation profiling by high throughput sequencing"],"species":["Coturnix japonica"],"pubmed_authors":["Céline Vandecasteele","Frédérique Pitel"],"additional_accession":[]},"is_claimable":false,"name":"DNA  methylation profiles of quail blood cells by whole-genome bisulfite and Oxford Nanopore sequencing","description":"Whole Genome Bisulfite Sequencing (WGBS) has been the gold standard DNA methylation mapping and quantification for over a decade. Oxford Nanopore Technologies (ONT) sequencing directly measures nucleotide modifications. In this study, we have compared DNA methylation levels (5-methylcytosine) at CpG sites in the quail genome using WGBS and ONT. Samples were collected to investigate transgenerational DNA methylation changes in Japanese quail following ancestral exposure to a phytoestrogen. Blood samples from 24 third-generation (G3) individuals—descendants of either treated or untreated ancestors—were sequenced after bisulfite conversion. Both methods revealed broadly consistent methylation patterns. ONT reads covered more CpG sites and detected a higher number of differentially methylated cytosines (DMCs). Principal component analyses showed that both sex and ancestral treatment groups accounted for a portion of the observed epigenetic variation, for both technologies. Strong concordance between WGBS and ONT results supports the reliability of ONT sequencing for epigenomic research, including in quails. These data pave the way for further investigation into whether genistein induces epigenetic changes for several generations.","dates":{"release":"2026-04-07T00:00:00Z","modification":"2026-05-21T14:30:29.852Z","creation":"2026-04-07T14:10:18.416Z"},"accession":"E-MTAB-16868","cross_references":{"ENA":["ERP139936","ERP155864"],"EFO":["EFO_0002944","EFO_0004170","EFO_0002761","EFO_0005518","EFO_0003816","EFO_0004184"]}}