{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Rene Ketting"],"organism":["Caenorhabditis elegans"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15751"],"description":["This study employed an unbiased forward genetic screen to identify suppressors of the maternal-effect lethal phenotype caused by tost-1 dysfunction in Caenorhabditis elegans. The temperature-sensitive tost-1(xf196 ts) mutant exhibits complete embryonic lethality at the restrictive temperature (25°C), providing a robust screening platform. Following EMS mutagenesis of L4 larvae, we screened F2 progeny for viable individuals at 25°C, indicating suppression of the lethal phenotype. After backcrossing to confirm heritability, we performed whole genome sequencing on 21 suppressor strains to identify causative mutations. Additionally we also sequenced the strain used for the mutagenesis as the reference genome."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Library Construction - Libraries were prepared using NEBNext Ultra II DNA Library Prep Kit starting with 10 ng fragmented DNA and 9 PCR cycles. Libraries were profiled on 2100 Bioanalyzer (Agilent) and quantified using Qubit 1× dsDNA HS Assay Kit. All 22 samples were pooled equimolarly and sequenced on NextSeq2000 P2 FC (2×100 cycles plus 2×8 index cycles).","Sample Collection - After confirmation of suppressor phenotypes and backcrossing to the parental tost-1(xf196 ts) strain, suppressor strains were expanded for genomic DNA extraction. Mixed-stage worm populations were cultured on 90 mm NGM plates seeded with E. coli OP50 for one generation, then transferred to 150 mm plates for an additional generation to increase biomass. Worms were harvested by washing plates with M9 buffer, collected by centrifugation, and washed multiple times to remove bacteria. Worm pellets were processed immediately for DNA extraction using the Gentra Puregene Tissue Kit (Qiagen) following the manufacturer's protocol for animal tissues. DNA concentration and purity were assessed by spectrophotometry before proceeding to library preparation.","Sequencing - Libraries were sequenced on NextSeq2000 (100 bp paired-end). Reads were adapter-trimmed using Cutadapt (v4.4) and mapped to C. elegans genome (WBcel235) using BWA-MEM2 (v2.2.1). Duplicates were removed with Picard (v3.0.0) and tracks generated using bamCoverage (v3.5.1). Variants were called using GATK HaplotypeCaller (v4.4.0.0) and filtered for SNPs, indels, and mixed variants. Additional filtering included variants present in TOST sample (background control), standard quality filters, and minimum depth of 6 using vcftools (v0.1.16). Variants were classified as homozygous/heterozygous using GATK tools, converted to MAF format, and annotated using VEP (v110.1). Expected G→A and C→T transitions were selected using bcftools (v1.17), and filtered VCF files were converted to XLSX format using R/Bioconductor packages.","Nucleic Acid Extraction - Genomic DNA was extracted using Gentra Puregene Tissue Kit (Qiagen). DNA (1.5 μg) was diluted to 55 μl in TE buffer and fragmented using Covaris S2 sonicator (Intensity: 5; Duty Cycle: 10%; Cycles/burst: 200; Time: 120 s; 2 cycles). Fragmented DNA was analysed on TapeStation with High Sensitivity D1000 ScreenTape."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - The variants were called using GATK Haplotypecaller (v4.4.0.0) and subsequently filtered using GATK's SelectVariants tool for SNPs, Indels and mixed variants. The identified variants were then filtered for variants present in the TOST sample using vcftools. The TOST sample serves as the background of the used strain towards the reference strain.  Additional filtering was done for standard quality and a minimal depth of 6 (parameter \\\"--filter  +/d=6\\\") by vcftools.  The variants were then classified as homozygous and heterozygous calls using GATKs SelectVariants and VariantFilteration tools.  Afterwards vcf files were converted to maf ( v1.6.21)  and annotated using VEP (v110.1).   The expected transitions from G to A and C to T were selected, using bcftools (v1.17). The filtered vcf files for homozygous calls were then converted to XLSX format using R/Bioconductor (v3.16) and a range of R/Bioconductor packages (Maftools v2.16, VariantAnnotation v1.46, openxlsx v4.2.5.2,tidyr  v1.3.0,purrr  v1.0.1, dplyr  v1.1.2 , tibble  v3.2.1).  The uploaded processed data files do contain the reference and quality filtered SNPs/Indels which were annotated by vep and converted into maf format.","Sequence Alignment - Reads were trimmed for sequencing adapters using Cutadapt (v4.4) and mapped against the C.elegans genome (WBcel235) using BWA-MEM2 (v2.2.1), read duplicates were removed using Picard (v3.0.0)."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["NextSeq 2000"],"study_type":["genotyping by high throughput sequencing"],"species":["Caenorhabditis elegans"],"pubmed_authors":["Rene Ketting"],"additional_accession":[]},"is_claimable":false,"name":"Whole genome sequencing of EMS-induced suppressors of tost-1 maternal-effect lethality in Caenorhabditis elegans","description":"This study employed an unbiased forward genetic screen to identify suppressors of the maternal-effect lethal phenotype caused by tost-1 dysfunction in Caenorhabditis elegans. The temperature-sensitive tost-1(xf196 ts) mutant exhibits complete embryonic lethality at the restrictive temperature (25°C), providing a robust screening platform. Following EMS mutagenesis of L4 larvae, we screened F2 progeny for viable individuals at 25°C, indicating suppression of the lethal phenotype. After backcrossing to confirm heritability, we performed whole genome sequencing on 21 suppressor strains to identify causative mutations. Additionally we also sequenced the strain used for the mutagenesis as the reference genome.","dates":{"release":"2025-11-11T00:00:00Z","modification":"2026-05-27T17:02:29.111Z","creation":"2025-10-16T11:09:37.797Z"},"accession":"E-MTAB-15751","cross_references":{"ENA":["ERP182290"],"EFO":["EFO_0002944","EFO_0004170","EFO_0002771","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0004184"]}}