2.2 Sequencing, Assembly, Annotation and Bioinformatic Analyses
Three complete mitogenomes were sequenced using next-generation
sequencing (NGS) on an Illumina HiSeq 2000 platform (Biomarker
Technologies, Beijing). The raw paired reads were retrieved and
quality-trimmed using CLC Genomics Workbench v10.0 (CLC Bio, Aarhus,
Denmark) with default parameters. The clean paired reads were then used
for mitogenome reconstruction using MITObim v1.7 software (Hahn,
Bachmann, & Chevreux, 2013) with default parameters and the mitogenome
of Ampittia dioscorides (KM102732) (Qin, Yang, Hou, & Li, 2017)
as the reference. Annotation of the mitogenomes and comparative analyses
were conducted following the methodology outlined above. The various
genomic features were annotated using Geneious 8.1.3 (Biomatters,
Auckland, New Zealand) and referenced to the complete mitogenome
sequence of A. dioscorides . Protein-coding genes (PCGs) were
determined by finding the ORFs based on the invertebrate codon table
(codon Table 5) and RNAs (tRNAs and rRNAs) were identified using MITOS
Web Server (Bernt et al., 2013). Transfer RNAs were manually plotted
according to the secondary structure predicted by MITOS, using Adobe
Illustrator CS5. Finally, all genes were visually inspected against the
reference mitogenome in Geneious. Nucleotide composition, codon usage,
comparative mitogenomic architecture tables for the three mitogenomes,
and data used to plot RSCU (relative synonymous codon usage) figures
were all calculated and created using PhyloSuite (D. Zhang et al.,
2020). The AT-skew and GC-skew were computed according to the following
formulas: AT-skew=[A−T]/[A+T] and GC-skew=[G−C]/[G+C]
(Perna & Kocher, 1995). The three newly-sequenced mitogenome sequences
of Aeromachini (Amipittia virgata , Halpe nephele andOnryza maga ) have been uploaded onto GenBank with the accession
number MW288057, MW288058 and MW288059, respectively.