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| Status |
Public on Apr 09, 2018 |
| Title |
P2-SSP-4 |
| Sample type |
SRA |
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| Source name |
colon
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| Organism |
Homo sapiens |
| Characteristics |
tissue: colon
tissue: Sessile serrated polyp biopsy from patient #2
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The characterized biopsies were subjected to sample isolation from the affected areas of the blocks using 1mm diameter disposable biopsy punches (Miltex). The QIAamp DNA FFPE Tissue kit (Qiagen) was used to extract DNA from FFPE tissues. The Qiagen AllPrep kit was used to extract both DNA and RNA from frozen O.C.T. embedded tissues. DNeasy Blood & Tissue kit (Qiagen) was used to extract DNA from blood samples. Library construction for exome sequencing was performed using Agilent Technologies SureSelect XT Reagent Kit, HSQ as described below. Briefly, 1-3 µg genomic DNA was sheared using Covaris S2 Focused-ultrasonicator, in a 130 µl volume, with 5.0 intensity, 10% duty cycle, 200 cycles per burst, and 6:00 minutes treatment time. Sheared DNA was purified using QIAquick PCR Purification kit (Qiagen).
Using a combination of enzymes with filling and exonuclease activity, the DNA fragments became blunt ended. To prepare the fragments for accepting adaptors and to block concatamerization, an A overhang was added to the 3’ end of the fragments, followed by ligation to adaptors with T overhangs. Adaptor-ligated fragments were amplified using 6 cycle PCR and followed by PCR product purification using Agencourt AMPure XP beads. Concentrations and fragment size ranges of the libraries were measured using NanoDrop spectrophotometer and D1K ScreenTape assay in the Agilent 2200 TapeStation system. Exome capture was performed on about 750 ng of the amplified libraries via biotinylated RNA bait molecules of SureSelect Human All Exon 50Mb (Agilent). The captured molecules were purified using Dynabeads MyOne Streptavidin T1, and index tagged in 10 PCR cycles. The amplified exome-enriched fragments were purified on Agencourt AMPure XP beads. Concentration and size measurements were done using Invitrogen Qubit dsDNA HS Assay and High Sensitivity D1K assay. The KapaBiosystems Kapa Library Quant kit was used to quantitate the concentration of adaptor ligated libraries. The captured libraries were sequenced using HiSeq 2000 101 cycle paired-end sequencing.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
whole exome-DNA
9482x1
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| Data processing |
Library strategy: Exome-seq
Reads were aligned to the genome with Novalign (v2.08.01) using default settings
SAMtools (v0.1.18) was used to create mpileup files from the alignments
VaScan (v2.3.5) was used to identify mutations.
Genome_build: hg19
Supplementary_files_format_and_content: VCF files
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| Submission date |
Feb 13, 2018 |
| Last update date |
Apr 09, 2018 |
| Contact name |
David A Jones |
| Organization name |
Oklahama medical research foundation
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| Department |
Functional and chemical genomics
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| Street address |
825 NE 13th st
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| City |
Oklahama city |
| ZIP/Postal code |
73104 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE110535 |
Active BRAF-V600E is the key player in generation of a sessile serrated polyp-specific DNA methylation profile (Exome-seq data set) |
| GSE110538 |
Active BRAF-V600E is the key player in generation of a sessile serrated polyp-specific DNA methylation profile |
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| Relations |
| BioSample |
SAMN08524397 |
| SRA |
SRX3688963 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2995352_9482X1.vcf.gz |
16.0 Mb |
(ftp)(http) |
VCF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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