introduction to the emerald dataset ron peterson anne bergstrom lucas, agilent jean lozach,...
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Introduction to the EMERALD Dataset
Ron Peterson Anne Bergstrom Lucas, Agilent Jean Lozach, Illumina Marc Salit, NIST Russ Wolfinger, SAS Walter Liggett, NIST Jean Thierry-Mieg, NCBI DanielleThierry-Mieg, NCBI
2 EMERALD dataset introduction
MicroArray Quality Control
Shippy, R. et al, Nature Biotechnology - 24, 1123 - 1131 (2006)
Titration working Group
Ambion Human Brain RNA Stratagene Universal Human RNA
3 EMERALD dataset introduction
MAQC Phase II – Conduct a new titration experiment
Agilent performed a one color analysis of the phase I material.
Added more intermediate titrations to a total of 19 samples.
Total of 80 samples processed
Experiment split up and performed over three days
First day used a mixture of reagent kits. Second day fresh kit. Third day reagents from colleague.
Ambion Brain HUR
100 0
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4 EMERALD dataset introduction
Evaluation of New Agilent Titration.
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Clustering of the arrays by amplify-label date. 3/2, 3/6, 4/10, 4/12
5 EMERALD dataset introduction
Sample information for Biological vs Technical variation study.
Animal: Rattus norvegicusStrain: Sprague Dawley Crl:CD(SD)Age: 7-8 weeksSex: Male Treatment: 20% propylene glycol/80%/lactic acid containing
4.3% mannitol, pH 4.0Duration : intravenous, once per week for 13 weeksNumber: 6
A 100% Liver 1A, 2A, 3A, 4A, 5A, 6A
B 75% Liver, 25% Kidney 1B, 2B. 3B, 4B, 5B, 6B
C 25% Liver, 75% Kidney 1C, 2C, 3C, 4C, 5C, 6C
D 100% Kidney 1D, 2D, 3D, 4D, 5D, 6D
6 EMERALD dataset introduction
Study Design
Each Sample was performed in triplicate
• eg, 1-A-1, 1A-2, 1-A3, etc.
• Each sample was placed into a single well of a 96 well plate in a randomized pattern.
• Remaining wells were filled with samples made from pooling animals.- 1-3A, B, C, D & 4-6A, B, C, D; single samples.
- 1-6A, B, C, D; each sample repeated 4 times on plate.
3 duplicate plates were produced and one plate was processed on;
• Affymetrix Rat Genome U133 plus 2.0 arrays
• Agilent Whole Rat Genome Oligo Microarray (4x44K) [G4131F]
• Illumina RatRef-12 v1 Expression BeadChip
7 EMERALD dataset introduction
Affymetrix Study design
Chip performed at the Novartis Institutes for Biomedical Research
96 well plate was processed on an Affymetrix GCAS robot.
96 chips were washed on 12 Fluidics Machines (48 chip lots).
48 chips lots were scanned on one of two Affymetrix Scanner.
• Technical variation parameters. - Sample plate location.
- Affymetrix chip lot.
- Fluidics station location.
- Scanner used.
- Day processed (8 of the 96 chips were processed on a different date by rehybridizing the hybridization mix on a new chip.
8 EMERALD dataset introduction
Agilent Study Design
Chips were processed at Agilent.
2 different technicians processed the arrays.
12 chips (48 arrays) were processed on 2 different days
A single scanner was used.
• Technical variation parameters- Technician.
- Day processed.
- Chip and reagent lots.
- Substrate.
- Starting total RNA amount (400 ng vs 200 ng).
9 EMERALD dataset introduction
Illumina Study Design
Chips were processed at Asuragen (service provider).
2 different technicians processed the arrays.
8 chips (96 arrays) were processed on 4 different days with 4 different kits.
A single scanner was used.
• Technical variation parameters- Technician.
- Day processed.
- Chip and reagent lots.
- Location on the chip.
- cRNA yield.
10 EMERALD dataset introduction
Data Access links
The data and supporting material are available from ArrayExpress
Affymetrix
• http://www.ebi.ac.uk/microarray-as/aer/result?queryFor=Experiment&eAccession=E-TABM-536
Agilent
• http://www.ebi.ac.uk/microarray-as/aer/result?queryFor=Experiment&eAccession=E-TABM-555
Illumina
• http://www.ebi.ac.uk/microarray-as/aer/result?queryFor=Experiment&eAccession=E-TABM-554
11 EMERALD dataset introduction
Current Plans of MAQC Phase II Titration Group
Jean and Danielle Thierry-Mieg (NCBI) have done a complete annotation of the rat genes in AceView and identified the alternative transcripts tested on all three rat arrays. http://www.aceview.org/index.html?rat
The mapping, available at ftp://ftp.ncbi.nlm.nih.gov/repository/acedb/rat, will be used to identify the groups of probes from the three array platforms testing the same transcripts and genes.
We will use this correspondence to contrast the performance of the arrays in their ability to identify biological and technical variation.