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, (2006) Titration working Group Ambion Human Brain RNAStratagene 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 BrainHUR

4 EMERALD dataset introduction Evaluation of New Agilent Titration Discriminate Coordinate Discriminate Coordinate 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 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 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 as/aer/result?queryFor=Experiment&eAccession=E-TABM-536  Agilent as/aer/result?queryFor=Experiment&eAccession=E-TABM-555  Illumina 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.  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.