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238 Abascal, Zardoya, and Posada4. Examples4.1. Characterizationof the Genetic Code ofSpeleonectestulumensis(Crustacea,Remipedia)The mt-genome of the arthropod species of Speleonectes tulumensis(16) is already deposited in GenBank and it is annotated at themoment of writing this chapter as having the InvertebrateMitochondrial Genetic Code (translation table number 5). TheNCBI taxonomic identifier is 84346, which we will use as inputfor GenDecoder (http://darwin.uvigo.es/software/gendecoder.html)leaving all other options with the default settings (alignmentpositions either with Shannon entropy values higher than 2.0or with more than a 20% of gaps are filtered out).After a while, not more than a couple of minutes, the output ofGenDecoder is printed out to the browser window. Some informationreferred to the alignments, the number of positions filtered,and the codon-usage is displayed first. Alignments can beinspected with the help of the program Jalview (15) by clicking inthe ‘‘get alignment’’ link next to the gene names. The main resultof GenDecoder is at the end of the page (Fig. 11.3a). Twopredictions (highlighted in red) are different from the annotationsin GenBank:(1) The AGC codon is predicted as Gly instead of Ser. TheAGC codon occurs 13 times in the mt-genome of S. tulumensis,butonly three instances occur at alignment positions below the defaultconservancy threshold. Hence, this prediction is based on threecodon occurrences only. In the ‘‘Freq-aa’’ line we can see that thefrequency of Gly is only around 0.3 (previously, in the results page,it was indicated that the frequency is exactly 0.32), and in the line‘‘Diff-freq’’ it is indicated that this frequency is only about 0.1 largerthan the frequency of the expected amino acid (Ser, 0.24). Byclicking on the red ‘‘g’’ (see Note 3) we can see the three alignmentpositions at which AGC appear (Fig. 11.3b): twice in the COX1alignment (positions 56 and 167) and once in the ND4 alignment(position 174). The poor support for the AGC assignment (lownumber of codons and low frequency of the predicted amino acid)suggests that this is an unreliable prediction. In fact, if we runGenDecoder with a softer threshold to include ‘‘variable sites (S