文本完整讲义印度昌迪加尔市微生物技术研究所生物信息学课件anal_marray.ppt

Analysis of Microarray Data Analysis of images Preprocessing of gene expression data Normalization of data Subtraction of Background Noise Global/local Normalization House keeping genes (or same gene) Expression in ratio (test/references) in log Differential Gene expression Repeats and calculate significance (t-test) Significance of fold used statistical method Clustering Supervised/Unsupervised (Hierarchical, K-means, SOM) Prediction or Supervised Machine Learnning (SVM) Technical Images from scanner Resolution standard 10?m [currently, max 5?m] 100?m spot on chip = 10 pixels in diameter Image format TIFF (tagged image file format) 16 bit (65’536 levels of grey) 1cm x 1cm image at 16 bit = 2Mb (uncompressed) other formats exist e.g.. SCN (used at Stanford University) Separate image for each fluorescent sample channel 1, channel 2, etc. Images in analysis software The two 16-bit images (Cy3, Cy5) are compressed into 8-bit images Display fluorescence intensities for both wavelengths using a 24-bit RGB overlay image RGB image : Blue values (B) are set to 0 Red values (R) are used for Cy5 intensities Green values (G) are used for Cy3 intensities Qualitative representation of results Images : examples Processing of images Addressing or gridding Assigning coordinates to each of the spots Segmentation Classification of pixels either as foreground or as background Intensity determination for each spot Foreground fluorescence intensity pairs (R, G) Background intensities Quality measures Background intensity Spot’s measured intensity includes a contribution of non-specific hybridization and other chemicals on the glass Fluorescence from regions not occupied by DNA should by different from regions occupied by DNA - one solution is to use local negative controls (spotted DNA that should not hybridize) Different background methods : Local background Morphological opening Constant background No adjustment Local background Focusing on small regions surrounding the