Briefly, after the genomic DNA was randomly broken into fragments using a Covairs ultrasonic breaker, a PE400 genomic DNA library was constructed based on the Whole Genome Shotgun (WGS) strategy and sequenced utilizing an Illumina NovaSeq 6000 instrument (Illumina, USA). More than 2.58 Gb of raw reads were obtained, and at least 1.67 Gb of clean reads were obtained from the raw reads after removing adaptor sequences using AdapterRemoval v2 [25 (link)] and filtering low-quality sequences using SOAPdenovo2 [26 (link)]. The obtained clean data were assembled using A5-miseq v20150522 [27 (link)] and SPAdesv3.9.0 [28 (link)] to construct contigs and scaffolds. A colinear analysis for splicing sequences was conducted using mummer v3.1 [29 (link)] and the complete mitogenome sequence was then revised and verified using pilon v1.18 [30 (link)]. All these procedures were performed by Shanghai Personal Biotechnology Co., Ltd., China.
The locations of protein-coding genes (PCGs) and rRNA genes were preliminarily predicted using GeSeq [31 (link)] and MITOS [32 (link)], and the precise locations were confirmed using related Hirudinea mitogenomes from GenBank. The initiation and termination codons were identified using ORF finder and Blastn of NCBI, according to their alignment with other related species, and the rearrangements of mitochondrial genes in the Hirudinea species were examined simply by observation. MITOS [32 (link)] was used to predict and annotate the locations and secondary structures of tRNA genes, and these were then visualized using the online software TBI-forna (http://rna.tbi.univie.ac.at/forna/ , accessed on 4 January 2022). The nucleotide composition and the relative synonymous codon usage (RSCU) were determined using MEGA 7 [33 (link)].
AT skew = (A − T)/(A + T) and GC skew = (G − C)/(G + C) were analyzed to describe base composition [34 (link)]. A graphical diagram of the complete mitogenome was drawn using the online mitochondrial visualization tool mtviz (http://pacosy.informatik.uni-leipzig.de/mtviz/mtviz , accessed on 4 January 2022). Finally, the complete mitochondrial DNA sequence was uploaded to the GenBank database with accession number OL779256.
The locations of protein-coding genes (PCGs) and rRNA genes were preliminarily predicted using GeSeq [31 (link)] and MITOS [32 (link)], and the precise locations were confirmed using related Hirudinea mitogenomes from GenBank. The initiation and termination codons were identified using ORF finder and Blastn of NCBI, according to their alignment with other related species, and the rearrangements of mitochondrial genes in the Hirudinea species were examined simply by observation. MITOS [32 (link)] was used to predict and annotate the locations and secondary structures of tRNA genes, and these were then visualized using the online software TBI-forna (
AT skew = (A − T)/(A + T) and GC skew = (G − C)/(G + C) were analyzed to describe base composition [34 (link)]. A graphical diagram of the complete mitogenome was drawn using the online mitochondrial visualization tool mtviz (
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