|
| Status |
Public on May 18, 2012 |
| Title |
Mt DNA 6 |
| Sample type |
genomic |
| |
|
| Source name |
43_SiRAN_3_Diploid_Ncalls
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Fibroblast cells
treatment: Cells were incubated with ITPA siRNA for 96hrs. Treatment with ITPA siRNA was for an unrelated biological question that was outside the scope of analysis of stretches of N calls.
|
| Treatment protocol |
Only three cells lines (Mt DNA 2, Mt DNA 4 and MtDNA 6) were manipulated, beyond described growth conditions. Cell lines were incubated with ITPA siRNA, to block metabolism of rogue nucleotides such as dITP and dXTP. Cellular DNA then was extracted after a 96h incubation period to give time for rogue nucleotides to accumulate. DNA was analysed alongside the other 13 samples.
|
| Growth protocol |
Eight fibroblasts cells recruited from patients with suspected metabolic diseases stored in liquid nitrogen at cell banks, Mater Adult Hospital (MHS), were recovered and cultured. Confluent fibroblast cell lines were received from the cell bank in 25cm2 NUNC vented flasks (Invitrogen). All fibroblast cell lines were genotyped for ITPA two common mutations (94C>A and IVS2+21 A>C), for analysis independent of this project. Ethics approval for the study was gained from the Matar Hospital. To maintain fibroblast cell cultures a complete media was used which contained: RPMI- 1640, without L-glutamine (Gibco BRL, Invitrogen, Australia), 10% foetal calf/bovine serum (FCS) (Gibco BRL, Australia), 100U/ml Penicillin, 100µg/ml Streptomycin, and 0.3g/ml L- glutamine (Invitrogen). Blood and bone marrow samples were extracted fresh and no growth conditions applied.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total DNA was extracted using a QIAamp DNA blood mini kit (Qiagen, Valencia, CA), the extractions performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
The entire mitochondrial genome (mtDNA) was amplified using three overlapping long PCR fragments using TaKaRa LA Taq (TaKaRa Biomedicals), with each reaction containing 25ng of genomic DNA. The three primers sets are described in the Affymetrix MitoChip v2.0 supporting documentation (GeneChip Human Mitochondrial Resequencing Array 2.0 Supporting Documentation). After target amplification, PCR products were purified using a High Pure PCR Product Purification Kit (Roche, Germany). PCRs were pooled, fragmented and end labelled according to Affymetrix GeneChip CustomSeq Resequenciong protocol (fragmentation and labeling enzymes and reagents were part of the Affymetrix GeneChip Resequencing Reagent Kit).
|
| |
|
| Hybridization protocol |
Following fragmentation, fragmented mtDNA were hybridized for 16 hours at 49°C with 60 rpm rotation in a hybridization mix solution. MitoChip v2.0 was then washed and stained using the Affymetrix fluidics station 450 (Mini_DNAArray_WS5_450).
|
| Scan protocol |
Data was acquired on a 7G GeneChip Scanner 3000 and data extraction performed in GCOS version 1.4 Nucleotide calls made using GSEQ v 4.1.
|
| Description |
Total DNA (nuclear and mitochondrial)
Cells were incubated with ITPA siRNA for 96hrs. Treatment with ITPA siRNA was for an unrelated biological question that was outside the scope of analysis of stretches of N calls. Sample contained at least one stretch of N calls.
|
| Data processing |
The Affymetrix algorithm parameter settings recommended to achieve optimal performance were used to analyze the mitochondrial sequences, with ‘diploid’ selected as the genome model to enable the detection of heteroplasmy. A Quality Score Threshold (QST) of 3 provided the highest performance in terms of overall base calling accuracy and call rates by the GSEQ software. All other parameters used by the algorithm were also fixed for all samples: No Signal Threshold= 2, Weak Signal Fold Threshold= 20, Large SNR Threshold= 20, Min Fraction of Calls of Samples = 0.5, Trace Threshold = 1, Sequence Profile Threshold = -0.175.
additional results files (available on the Series Record): GSEQ results.txt, sPROFILER results.txt
GSEQ results.txt = Summary data from GSEQ using settings described in "data processing".
sPROFILER.txt = Summary data where data from GSEQ base calls was further analysed using sPROFILER (strand-specific PRObe cell intensity comparison for FILtERing- PMID20146813). This is a novel and free algorithm developed by Kothiyal et al 2010 for improving array call rates using MATLAB over GSEQ call rates, based on intensity signature.
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| |
|
| Submission date |
May 26, 2011 |
| Last update date |
May 18, 2012 |
| Contact name |
Gareth Price |
| E-mail(s) |
nscalehome@gmail.com
|
| Phone |
61413289611
|
| Organization name |
Queensland Facility for Advanced Bioinformatics
|
| Street address |
Carmody Road
|
| City |
Brisbane |
| State/province |
Queensland |
| ZIP/Postal code |
4072 |
| Country |
Australia |
| |
|
| Platform ID |
GPL10983 |
| Series (1) |
| GSE29550 |
Insights into N-calls of mitochondrial DNA sequencing using MitoChip v2.0 |
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