Computational protocol: Complete mitochondrial genomes of Trisidos kiyoni and Potiarca pilula: Varied mitochondrial genome size and highly rearranged gene order in Arcidae

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[…] All sequence data were analysed and arranged to create the full genomes using the Seqman program from DNASTAR (http://www.DNASTAR.com). The protein coding genes were analyzed with ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and BLASTx using the invertebrate mitochondrial genetic code. The tRNA genes were identified by ARWEN and DOGMA using the mito/chloroplast or invertebrate genetic code and the default search mode. The rRNA genes were identified by their similarity to published gene sequences and by using BLAST searches (http://www.ncbi.nlm.nih.gov/BLAST/).The base composition and skewness analyses were performed and compared between T. kiyoni and P. pilula genomes, as well as the other four Arcidae genomes (S. broughtonii (46,985 bp), S. kagoshimensis (46,713 bp), T. granosa (31,589 bp) and A. vellicata (34,147 bp)). The A + T content values were computed using Editseq program from DNASTAR. The GC and AT skews described strand bias were calculated according to the formulae by Perna and Kocher, AT skew = (A − T)/(A + T); GC skew = (G − C)/(G + C), where A, T, G and C are the occurrences of the four nucleotides. The codon usage of each PCG were calculated using MEGA 5. The ratios of nonsynonymous and synonymous substitutions rates (Ka/Ks) were estimated based on the Maximum-Likelihood (ML) method using KaKs_Calculator 2.0 with the YN model.The whole mitogenome sequence was tested for potentially tandem repeats by Tandem Repeats Finder 4.0. Prediction of potential secondary structure was performed by the online version of the mfold software, version 3.2, applying default settings. When multiple secondary structures were possible, the most stable (lowest free energy (ΔG)) was used.The gene map of the T. kiyoni and P. pilula mitogenomes were generated with the program CGView. The two mitochondrial genomes have been deposited in the GenBank database under the accession numbers KU975161 for T. kiyoni and KU975162 for P. pilula.Predicted lengths of gene products and mitogenome sizes for up to 278 molluscs (see ). The statistical analysis was performed by using IBM SPSS Statistics 19 with Spearman rank correlations, as this test makes no assumption about the distribution of the data. [...] Along with mitochondrial genome sequence of T. kiyoni and P. pilula, all currently available mitochondrial genomes from Arcidae, including S. broughtonii (AB729113), S. kagoshimensis (KF750628), T. granosa (KJ607173) and A. vellicata (KP954700), were used in phylogenetic analysis.The phylogenetic relationships were built based on the nucleotide sequences of 12 PCGs. Crassostrea gigas (AF177226) and Crassostrea hongkongensis (EU266073) from the family Ostreidae was used as outgroup. The twelve-partitioned nucleotide sequences of protein coding genes were aligned with MAFFT based on their nucleotide sequences using default settings. The final nucleotide sequences of each gene were then concatenated into single contigs (6719 bp) for phylogenetic analyses. The best-fit nucleotide substitution models for each data partitions were selected by jModelTest. We employed ML in RAxML Black-Box webserver (http://phylobench.vital-it.ch/raxml-bb/index.php) with GTR + G substitution model to each partition. For the ML analysis, 1000 bootstraps were used to estimate the node reliability. […]

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