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| Status |
Public on Apr 26, 2012 |
| Title |
ICF patient |
| Sample type |
SRA |
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| Source name |
Blood, ICF patient
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| Organism |
Homo sapiens |
| Characteristics |
tissue: blood
cell type: EBV immortalized B cells
disease status: immunodeficiency, centromere instability and facial anomalies (ICF) syndrome
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| Treatment protocol |
B cells of a healthy donor and ICF patient were immortalized by Epstein-Barr-Virus (EBV).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA were extracted using phenol:chloroform:isoamylalcohol (Sigma).
For normal BS-seq library construction, 10μg genomic DNA was fragmented using a Covaris sonication system (Covaris S2). Following fragmentation, libraries were constructed using the Illumina Paired-End protocol consisting of end repair, <A> base addition and methylated-adaptor ligation. Ligated DNA was bisulfite converted using the EZ DNA Methylation-Gold kit (ZYMO). Different insert sizes were excised from the same lane of a 2% TAE agarose gel. Normally, three bands are excised corresponding to DNA insert sizes of 80-100 bp, 100-120 bp to 120-150bp. Products were purified by using the QIAquick Gel Extraction kit (Qiagen) and amplified by PCR. PCR was carried out in a final reaction volume of 50μl consisting of 20μl purified DNA, 4μl 2.5 mM dNTP, 5 μl 10X buffer, 0.5 μl JumpStart™ Taq DNA polymerase, 2μl 10uM PCR primers and 37.5 μl ΜltraPure TM Water and the following thermal cycling program: 94ºC 30 s, 10 cycles of 94ºC 30 s, 60ºC 30 s, 72ºC 30 s then prolong with 1 min at 72ºC. PCR products were sequenced with a HiSeq 2000. The alignment was performed using GEM.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Jesus4
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| Data processing |
Illumina Casava1.7 software was used for basecalling.
Read mapping: Two reference sequences were prepared based on the hg19 human reference; referenceC2T had the C residues replaced by T's, and referenceG2A had the G’s replaced by A's. Two sets of reads were generated in the same way; setC2T with the C’s replaced by T's, and setG2A with the G's replaced by A's. Both sets of reads were aligned to each of the two references, producing four sets of mappings. This mapping strategy allowed reads to be aligned irrespective of the methylation status of any C residues.
The alignment was performed using the GEM alignment software allowing up to 4 mismatches in high quality bases (bases with quality scores > 25). Uniquely mapping read pairs were used to generate an empirical distribution of insert sizes of each of the libraries, and this information was used to select read pairs with unique compatible mappings.
The bisulfite conversion followed by a PCR step generates 4 different strands; the original + and - strands along with their complements. Reads derived from the original + strand (and its complement) will align to referenceC2T, while reads from the original - strand (and complement) will align to referenceG2A. Both members of a read pair are derived from the same original strand, and >99% of mapped reads could have the original strand assigned unambiguously. By keeping the information on the original strand origin of each read pair, the methylation status of C residues on either of the two original strands can be inferred along with the underlying genotypes, because any conversion due to the bisulphite treatment will only be seen on read pairs deriving from one of the two original strands.
Genome_build: hg19
Supplementary_files_format_and_content: Methylation calls files for C's in the bsmap alignment files were generated using methratio.py.
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| Submission date |
Apr 25, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Manel Esteller |
| Organization name |
IDIBELL
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| Department |
PEBC
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| Lab |
Cancer Epigenetics
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| Street address |
Hospital Duran i Reynals Av. Gran Via s/n km, 2.7
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| City |
L'Hospitalet de Llobregat |
| State/province |
Barcelona |
| ZIP/Postal code |
08908 |
| Country |
Spain |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE37578 |
Epigenetic alterations of a DNMT3B-mutant ICF patient at base-pair resolution |
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| Relations |
| SRA |
SRX144337 |
| BioSample |
SAMN00989114 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM922329_Jesus4.bs-call.basecall.txt.gz |
3.9 Gb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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