1 Bioinformática Posgrado

2 Image Analysis` Background Subtraction Normalization Summarization Transformation Data Processing Background Detection & Subtraction a) Filtering Microarray Image Scanning Spot Detection Intensity Value Affymetrix Two-dyes b) Image Analysis and Background Subtraction c) Transformation Between Within d) A=log2(R*G)/2 M=log2(R/G) Normalization

3  UID  IA  IB  R  C  MR  Print-tip Normalization  MC  Print-tip Normalization  SR  SC  FlagA  FlagB  SA  SF  QC  QCA  QCB  BkgA  BkgB  SDA  SDB  SDBkgA  SDBkgB  MedA  MedB  MNA  Signal Ch. A = Cy3 [Green]  MNB  Signal Ch. B = Cy5 [Red]  MedBkgA  Background Ch. A  MedBkgB  Background Ch. B  X  Y  PValueA  PValueB

4  Block  Print-tip Normalization  Column  Row  Name  ID  X  Y  Dia.  F635 Median  F635 Mean  F635 SD  B635 Median  B635 Mean  B635 SD  % > B635 + 1 SD  % > B635 + 2 SD  F635 % Sat.  F532 Median  F532 Mean  F532 SD  B532 Median  B532 Mean  B532 SD  % > B532 + 1 SD  % > B532 + 2 SD  F532 % Sat.  Ratio of Medians  Ratio of Means  Median of Ratios  Mean of Ratios  Ratios SD  Rgn Ratio  Rgn R²  F Pixels  B Pixels  Sum of Medians  Sum of Means  Log Ratio  Flags  Normalize  F1 Median - B1  F2 Median - B2  F1 Mean - B1  Signal - Background  F2 Mean - B2  Signal - Background  SNR 1  F1 Total Intensity  Index  "User Defined" http://www.moleculardevices.com/pages/software/gn_genepix_file_formats.html#gpr

5  www.gepas.org www.gepas.org  Casi cualquier Microarreglo  Excepto SpotFinder!  MIDAS TM4

6  http://www.tm4.org/mi das.html http://www.tm4.org/mi das.html  (1) Project  New  (2) Read Data  Single Data File  Especifique su archivo.MEV  (3) Operations  LocFit Normalization (Lowess)  “Loc” – Local = por print tip, Lowess o Loess es la normalización de MA

7  (4) Write Data  Desactive las casillas “virtual”  (5) Pulse Execution  Esto ejecuta la normalización y escribe algunos archivos.  Escribe 2 carpetas llamadas “raw” (datos crudos u originales) y “lowess” (resultado de la normalización)

8  Visualice los resultados en pestaña “Investigation”  De click con el botón derecho en cada rubro y use “Plot” tanto en raw como en lowess