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| Status |
Public on Feb 01, 2010 |
| Title |
Enrich1_50 |
| Sample type |
SRA |
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| Source name |
blood
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| Organism |
Homo sapiens |
| Characteristics |
tissue: blood
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was fragmented for 6 minutes using a Covaris S2 sonicator (6 x 16 mm AFA fiber Tube, duty cycle: 20%, intensity: 5, cycles/burst: 200, frequency sweeping). After fragmentation, fragments were blunt-ended and phosphorylated at the 5' end using End-it Kit (Epicentre) according to the manufacturer’s instructions, followed by ligation of double-stranded short adapters (adapter 1: pre-annealed duplex of 5'-CTA TGG GCA GTC GGT GAT-3' and 5'-ATC ACC GAC TGC CCA TAG TTT-3' and adapter 2: pre-annealed duplex of 5'-CGC CTT GGC CGT ACA GCA G-3' and 5'-GCT GTA CGG CCA AGG CG-3'; all oligo’s were acquired through Integrated DNA Technologies (Coralville, IA) and pre-annealing was done by mixing complementary oligonucleotides at 500 µM concentration and running on thermocycler with the following program: 95˚C for 3 min, 80˚C for 3 min, 70˚C for 3 min, 60˚C for 3 min, 50˚C for 3 min, 40˚C for 3 min and 4˚C hold). Ligation was performed using Quick ligation kit (New England Biolabs) with 1 µg of fragmented DNA, 750 nM adaptor 1 and adaptor 2, 150 µl of 2x Quick ligation buffer, and 5 µl Quick Ligase in a total volume of 300 µl. Samples were purified on Ampure beads (Agencourt) and run on a native 6% polyacrylamide gel. Fragments ranging from 150 to 175 bp were excised; the piece of gel containing fragments was shredded and dispersed into 400 µl of Platinum PCR Supermix with 750 nM of both amplification PCR primers ( provide sequence of amplification primers), 2.5 U of Pfu DNA polymerase (Stratagene) and 5 U Taq DNA polymerase (Bioline). Before ligation-mediated amplification, the PCR sample was incubated at 72˚C for 20 minutes in PCR mix to let the DNA diffuse from the gel and to perform nick translation on non-ligated 3'-ends. After 8 cycles of amplification, the library DNA was purified on Ampure beads and the quality was checked on a gel for the proper size range and the absence of adapter dimers and heterodimers. This library served as a stock for all subsequent hybridization experiments.
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| Library strategy |
WGS |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
AB SOLiD System 3.0 |
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| Description |
The SNP supplementary files have 384 known positions. The SNP_1_50_all file has all SNPs.
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| Data processing |
Sequencing reads were mapped against the reference genome (hg18 assembly, NCBI build 36) using the Maq package, which allows mapping in SOLiD color space corresponding to dinucleotide encoding of the sequenced DNA with following settings: number of maximum mismatches that can always be found -n 3, threshold on the sum of mismatching base qualities -e 150. Raw variant positions were called by the Maq package and filtered using custom scripts (available upon request). For stringent SNP calling we used the following filtering settings: 1) positions with lower than 20x and higher than 5000x coverage were excluded, 2) each of non-reference alleles had to be supported by at least 3 independent reads (as determined by different read start positions) separately on positive and negative strand with quality > 10, 3) the non-reference allele should account for at least 20% of the reads covering the polymorphic position, and 4) the ratio between + and – strand reads should be between 1/9 and 9. Positions that passed these filtering settings were considered as SNPs. A SNP was qualified as homozygous when the fraction of non-reference alleles was above 95% and heterozygous when the fraction of non-reference alleles was between 20% and 95%.
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| Submission date |
Oct 13, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Michal Mokry |
| E-mail(s) |
m.mokry@umcutrecht.nl
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| Organization name |
Wilhelmina Children's Hospital, University Medical Center Utrecht
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| Street address |
Lundlaan 6
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| City |
Utrecht |
| ZIP/Postal code |
3584 EA |
| Country |
Netherlands |
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| Platform ID |
GPL9442 |
| Series (1) |
| GSE18542 |
Highly efficient genomic enrichment of human exonic sequences through short-fragment libraries |
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| Relations |
| SRA |
SRX016325 |
| BioSample |
SAMN00008416 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM461684_Alignment_1_50.bed.gz |
77.6 Mb |
(ftp)(http) |
BED |
| GSM461684_SNPs_1_50.txt.gz |
7.4 Kb |
(ftp)(http) |
TXT |
| GSM461684_SNPs_1_50_all.txt.gz |
36.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
| Raw data are available in SRA |
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