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| Status |
Public on Dec 31, 2023 |
| Title |
RRBS, ALS, ALS75 |
| Sample type |
SRA |
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| Source name |
blood
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| Organism |
Homo sapiens |
| Characteristics |
tissue: blood
patient code: ALS75
analysis group: ALS
Sex: male
age (at_sampling): 59
age (at_als_onset): 57
disease: ALS
symptoms: lower and upper limb, bulbar not present
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA extraction from whole blood. We prepared aliquots of 1 ml frozen whole blood sample from ALS and control patients and extracted 200 μL of it using the Macherey-Nagel™ NucleoSpin™ Tissue Kit according to the manufacturer's protocol.
The premium RRBS kit of Diagenode was employed to carry out the RRBS protocol including NGS library preparation, as described (Veillard, AC., Datlinger, P., Laczik, M. et al. Diagenode® Premium RRBS technology: cost-effective DNA methylation mapping with superior coverage. Nat Methods 13, i–ii (2016). https://doi.org/10.1038/nmeth.f.391). Briefly, 100 ng of DNA was fragmented by the MspI restriction enzyme, which cleaves DNA without regard to the cytosine methylation state. Following adaptor ligation and size selection by Ampure beads, we pooled up to 6 samples together. These pooled samples underwent bisulfite treatment, purification, and PCR amplification, according to the manufacturer’s recommendations. The final RRBS libraries were quantified using the Qubit dsDNA HS Assay from Life Technologies, and the library's size distribution profile was assessed using Agilent Bioanalyzer 2100 capillary electrophoresis.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Sequencing was performed on Illumina HiSeq 2500 instrument with the 1x50 single-end sequencing mode.
Following an initial quality trimming, adapter sequences were removed from the raw reads using Trim Galore v0.6.10 (with Cutadapt v4.4) with parameters '--quality 20 --stringency 5 --length 35 --non_directional –rrbs'.
Bisulfite alignment to the GRCh38 reference genome and methylation calling was performed using Bismark v0.24.1 (with bowtie2 v2.3.5.1) with parameter '-N 1'. Alignment files were sorted with samtools v1.10.
Analysis of methylation data were performed using MethylKit v1.26.0 R package (with R v4.3.1). Bismark alignment data for CpG sites were read in and filtered using processBismarkAln() function with parameters 'read.context=CpG, mincov=10'.
Methylation data were filtered and normalized using filterByCoverage() function with parameters 'lo.count=10, hi.perc=99.9' and normalizeCoverage() function with parameter 'method=”median”'.
ALS vs Ctrl DMS (differentially methylated CpG sites) analysis was performed using the following functions: merging all sample data and filtering using unite() with the parameters 'min.per.group=4L, destrand=TRUE'; calculating differential methylation statistics using calculateDiffMeth() with default parameters'; DMS selection using getMethylDiff() with parameters 'difference=20, qvalue=0.01'.
ALS vs Ctrl DMR (differentially methylated regions) analysis was performed using the following functions: tiling the genome using tileMethylCounts() with parameters 'win.size=1000, step.size=1000, cov.bases=5'; merging all sample data and filtering using unite() with the parameters 'min.per.group=4L'; calculating differential methylation statistics using calculateDiffMeth() with default parameters; DMR selection using getMethylDiff() with parameters 'difference=20, qvalue=0.01'.
DMSs and DMRs were annotated with the overlapping genes (exon, intron, promoter, TSS) using the GRCh38.p13 annotation data.
Assembly: hg38
Supplementary files format and content: Raw CpG methylation data for each sample (txt)
Supplementary files format and content: DMS list with differential methylation statistics and annotation (tsv)
Supplementary files format and content: DMR list with differential methylation statistics and annotation (tsv)
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| Submission date |
Sep 06, 2023 |
| Last update date |
Dec 31, 2023 |
| Contact name |
Lorant Szekvolgyi |
| Organization name |
University of Debrecen
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| Department |
Department of Pharmacy
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| Lab |
Genome Architecture and Recombination Research Group
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| Street address |
Egyetem ter 1.
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| City |
Debrecen |
| ZIP/Postal code |
4032 |
| Country |
Hungary |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE242474 |
DNA methylome, R-loop and clinical exome profile of patients with sporadic Amyotrophic Lateral Sclerosis (RRBS) |
| GSE242475 |
DNA methylome, R-loop and clinical exome profiling of patients with sporadic Amyotrophic Lateral Sclerosis |
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| Relations |
| BioSample |
SAMN37304314 |
| SRA |
SRX21649979 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7764389_ALS75_CpG.txt.gz |
11.9 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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