2.2. Annotation of the Vischeria sp. CAUP Q 202 and Monodopsis sp. MarTras21 plastid genomes

An initial annotation of the two new plastome sequences was obtained using MFannot (http://megasun.bch.umontreal.ca/cgi-bin/mfannot/mfannotInterface.pl). Prediction of individual genes was checked by comparison to homologous sequences (primarily from other eustigmatophytes), which led us to revise the definition of the actual initiation codons of some of the genes. 5′ and 3′ ends of genes for non-coding RNAs (rRNAs and tRNAs) were likewise checked and adjusted by comparison to orthologous genes from other eustigmatophytes. Predicted intergenic regions were translated in all six frames and the conceptual translations were used as queries in blastp searches [32] against the non-redundant (nr) protein sequence database at the National Center for Biotechnology Information (NCBI; http://blast.ncbi.nlm.nih.gov/Blast.cgi). In parallel, the possible presence of protein-coding genes known to reside in previously sequenced eustigmatophyte plastid genomes, but not predicted by MFannot in the two newly sequenced genomes, was tested by tblastn searches against the respective plastid genome assemblies using the respective protein sequences from other eustigmatophytes. This led to the identification of some short genes missed by MFannot. A previously missed ssrA gene was identified in all eustigmatophyte plastid genomes thanks to the comparison of the plastid gene repertoire in different ochrophytes; its borders were delimited according to a published annotation of the gene in diatom plastomes. We also identified and incorporated into the annotation an intron-interrupted leu-tRNA gene that is conserved in eustigmatophyte plastomes [12] yet was missed by MFannot. Circular genome maps were generated with OGDraw v.1.2 [35]. A list of genes identified in plastomes of Vischeria sp. CAUP Q 202, Monodopsis sp. MarTras21 and selected other eustigmatophytes and ochrophytes is provided in the electronic supplementary material, table S1.