Summary of Affymetrix Microarray Data Analysis

1. Affymetrix Microarray Data

• CEL files: contain intensity values, higher intensity (transcript abundance) more active genes
• CDF (chip description file) files: specify the probe and probe set to which each cell belongs
• Terms:
  • Probe: oligonucleotides of 25 base (pair) length used to probe RNA targets (25 base sequence)
  • Probe pair: a unit composed of a perfect match (PM) and its mismatch (MM)
  • Probe pair set: PMs and MMs related to a common affyID (a group of probe pairs corresponds to a particular gene or a fraction of a gene. Some genes are represented by more than one probe set.)
  • affyID: an identification for a probe set (which can be a gene or a fraction of a gene) represented on the array

2. Microarray Experimental Designs

• Biological and technical replicates
• Pooling (biological averaging), blocking, randomized
• Sample size determination

3. Data Exploration

• MA plots
  • M values are log fold changes, M=log2(T/C)=log2(T)-log2(C)
  • A values are average log intensities between two arrays, A=(log2(T)+log2(C))/2
• Images, residual images
• Histograms, boxplots
• RNA degradation plots

4. Data Preprocessing

• Approaches: background correction, normalization, PM correction, and summarization
  • Background correction methods:
    • rma: robust multiarray average method (Irizarry et al. 2003)
    • mas: Affymetrix Microarray Suite background correction method (2002)
    • GCRMA: modified RMA to estimate nonspecific binding (Wu et al. 2004)
  • Normalization methods:
    • quantile, contrast and loess: discussed and compared by Bolstad et al. (2003)
    • constant (scaling): taken by Affymetrix, usually done after summarization
    • invariantset: used in the dChip software (Li and Wong 2001)
    • qspline: normalized by fitting splines to the quantiles (Workman et al. 2002).
  • PM correction methods:
    • mas: an ideal mismatch subtracted from PM (Affymetrix 2002)
    • pmonly: no adjustment to the PM values.
    • subtractmm: subtract MM from PM (Affymetrix MAS 4.0 1999)
  • Summarization methods:
    • avgdiff: the average (Affymetrix MAS 4.0 1999)
    • mas: Tukey biweight on log2(PM-CM) (Affymetrix MAS 5.0 2002)
    • liwong: model-based expression index (MBEI) (Li and Wong 2001), fitting the following multi-chip model to each probeset:
      • y_ij = theta_i * phi_j + epsilon_ij, where y_ij = PM_ij - MM_ij
      • y_ij = mu_i + theta_i * phi_j + epsilon_ij, where y_ij = PM_ij
    • medianpolish: used in the RMA expression summary (Irizarry et al. 2003). A multichip linear model is fit to data from each probeset
      • y_ij = alpha_i + beta_j + epsilon_ij, where y_ij are the background-adjusted, normalized, and log-transformed PM intensities
    • playerout: Lazaridis et al. (2002)

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