1. 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. 2 EMERALD dataset introduction MicroArray Quality Control Shippy, R. et al, Nature Biotechnology - 24, 1123 - 1131 (2006) Titration working Group Ambion Human Brain RNAStratagene Universal Human RNA

3. 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 BrainHUR 1000 99.50.5 991 955 9010 8020 7525 7030 6040 50 4060 3070 2575 2080 1090 595 199 0.599.5 0100

4. 4 EMERALD dataset introduction Evaluation of New Agilent Titration. -20-10010 Discriminate Coordinate 1 -15 -10 -5 0 5 10 Discriminate Coordinate 2 14 13 17 13 11 18 20 10 17 15 1 8 8 2 3 4 10 3 17 18 13 16 20 9 15 11 6 12 19 5 11 19 5 12 7 20 13 6 7 2 12 5 11 14 18 19 20 1 4 9 7 15 1 2 10 7 6 14 5 16 3 8 4 3 9 18 14 16 12 1 17 19 2 8 9 10 16 6 4 Clustering of the arrays by amplify-label date. 3/2, 3/6, 4/10, 4/12

5. 5 EMERALD dataset introduction Sample information for Biological vs Technical variation study. Animal: Rattus norvegicus Strain: Sprague Dawley Crl:CD(SD) Age: 7-8 weeks Sex: Male Treatment: 20% propylene glycol/80%/lactic acid containing 4.3% mannitol, pH 4.0 Duration : intravenous, once per week for 13 weeks Number: 6 A100% Liver1A, 2A, 3A, 4A, 5A, 6A B75% Liver, 25% Kidney1B, 2B. 3B, 4B, 5B, 6B C25% Liver, 75% Kidney1C, 2C, 3C, 4C, 5C, 6C D100% Kidney1D, 2D, 3D, 4D, 5D, 6D

6. 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. 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. 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. 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. 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. 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 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. ftp://ftp.ncbi.nlm.nih.gov/repository/acedb/rat  We will use this correspondence to contrast the performance of the arrays in their ability to identify biological and technical variation.