P. pacificus orphan genes are enriched in atypical codon usage. (A) For all P. pacificus genes with >100 codons, RSCU values were computed, and RSCU and GC3 deviation of a single gene from the genomewide average were plotted. Both values show a substantial correlation indicating that RSCU values are strongly biased by GC3 variation. (B) Correlation between GC3 and intronic GC content suggests that this bias is due to genomic variation in GC content and not specific for individual transcripts. (C) GC-normalization of RSCU values strongly reduces the correlation between deviation in GC3 and codon usage bias. (D) Cumulative distribution of P. pacificus orphan genes that were ranked by their deviation from the genomewide average of RSCU, GC-normalized RSCU and GC3 content. Genes with most atypical codon usage profiles show an enrichment in orphan genes (P = 0.0002), and the strongest enrichment is observed for the GC-normalized RSCU values.