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| Status |
Public on Nov 26, 2018 |
| Title |
control_3_blood (RRBS) |
| Sample type |
SRA |
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| Source name |
peripheral blood leukocytes
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| Organism |
Homo sapiens |
| Characteristics |
disease state: healthy control
cell type: peripheral blood leukocytes
genotype: control
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| Treatment protocol |
N/A
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| Growth protocol |
N/A
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA isolated with DNeasy Blood & Tissue Kit (QIAGEN) or Nucleon BACC2 Genomic DNA Extraction Kit (illustra) and quantified by Qubit (Invitrogen). DNA from mouse cortex samples were concentrated using Agencourt AMPure XP technology.
200ng of purified DNA samples were processed using the Ovation RRBS Methyl-Seq system kit according to instructions with the following modifications (NuGen Technologies). Briefly, unmethylated phage λ DNA 0.5ng (NEB) was spiked into each sample to allow assessment of bisulfite conversion efficiency. The methylation-insensitive restriction enzyme MspI was then used to digest the gDNA, and digested fragments were ligated to adapters. Adapter ligated fragments were then repaired before bisulfite conversion with the Qiagen Epitect fast bisulfite conversion kit. Bisulfite-treated adapter-ligated fragments were amplified by 9 cycles of PCR and purified using Agencourt RNAClean XP beads. Libraries were quantified using the Qubit dsDNA HS assay and assessed for size and quality using the Agilent Bioanalyzer DNA HS kit. Sequencing was performed using the NextSeq 500/550 high-output version 2 kit (75bp paired end reads) on the Illumina NextSeq 550 platform. As instructed for the NuGen RRBS kit, 12bp index reads were generated to sequence the Unique Molecular Identifiers (UMI) in addition to the index present in the adaptors. Libraries were combined into equimolar pools and run on a single flow cell. 10% PhiX control library was spiked in to facilitate sequencing by generating additional sequence diversity. Library preparation and sequencing performed by WTCRF, Edinburgh.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
NextSeq 550 |
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| Description |
C3
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| Data processing |
Raw Illumina sequencing output from the NextSeq were converted to paired FASTQ files without demultiplexing using ‘bcl2fastq’ and default settings (v2.17.1.14). These FASTQ files were then demultiplexed using custom python scripts considering indexes with perfect matches to the sample indexes. The different lanes for each sample were then combined.
Sequencing quality was assessed with FASTQC (v0.11.4). Low quality reads and remaining adaptors were removed using TrimGalore (v0.4.1, Settings: --adapter AGATCGGAAGAGC --adapter2 AAATCAAAAAAAC). NuGen adaptors contain extra diversity bases to facilitate sequencing. These were removed using the ‘trimRRBSdiversityAdaptCustomers.py’ Python script provided by NuGen (v1.11). The paired end reads were then aligned to the hg19 genome using Bismark (v 0.16.3 with Bowtie2 v2.2.6 with settings: -N 0 -L 20) before PCR duplicates were identified and removed using the 6bp UMIs present in the index reads and the ‘nudup.py’ Python script supplied by NuGen (v2.3). Aligned BAM files were processed to report coverage and number of methylated reads for each CpG observed. Forward and reverse strands were combined using Bismark’s methylation extractor and bismark2bedgraph modules with custom Python and AWK scripts.
Processed RRBS files were assessed for conversion efficiency based on the proportion of methylated reads mapping to the λ genome spike-in (>99.5% in all cases).
BigWigs were generated from RRBS data using CpGs with coverage ≥5
Genome_build: hg19
Supplementary_files_format_and_content: Processed coverage values for each CpG observed in sample. CpG position and total coverage (T) and methylated coverage (M) combining both strands. Columns = chr, start, end, T, M. Positions are 0-based, ie BED format.
Supplementary_files_format_and_content: bigWig files for each sample also supplied. Only contain CpGs with coverage >= 5 in that sample.
Raw sequencing reads are available through EGA (protected patient data): EGAS00001003232
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| Submission date |
Sep 27, 2018 |
| Last update date |
Nov 26, 2018 |
| Contact name |
Duncan Sproul |
| Organization name |
University of Edinburgh
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| Department |
MRC Human Genetics Unit
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| Street address |
Crewe Road South
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| City |
Edinburgh |
| State/province |
Mid Lothian |
| ZIP/Postal code |
EH4 2XU |
| Country |
United Kingdom |
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| Platform ID |
GPL21697 |
| Series (2) |
| GSE120554 |
Gain of function DNMT3A mutations cause microcephalic dwarfism and hypermethylation of Polycomb-regulated regions (human RRBS) |
| GSE120558 |
Gain of function DNMT3A mutations cause microcephalic dwarfism and hypermethylation of Polycomb-regulated regions |
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| Relations |
| BioSample |
SAMN10136763 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3402940_C3_RRBS.bigWig |
15.4 Mb |
(ftp)(http) |
BIGWIG |
| GSM3402940_C3_RRBS.cov.gz |
11.0 Mb |
(ftp)(http) |
COV |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
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