Williams, McCue, Schaeffer, & Wold, 2008), effectively alleviated the significant bias introduced by transcript length within controls and alcoholics (Fig. 11.5D). Utilizing RPKM values also blunted the relationship between GC content and computed expression values (Fig. 11.6D), although not to the same degree as the length of expressed biotypes. The effect of GC content on expression may be minimized with additional processing/normalization strategies (Hansen, Irizarry, & Wu, 2012; Risso, Schwartz, Sherlock, & Dudoit, 2011). At first glance, expression may appear nearly indistinguishable between alcoholics and controls (Figs. 11.5 and 11.6); however, detailed examination of RNA-Seq can expose biases that may affect expression estimates, and an appropriate normalization strategy is therefore crucial.
