De novo assembled mitogenome analysis of Trichuris trichiura from Korean individuals using nanopore-based long-read sequencing technology

Abstract

Knowledge about mitogenomes has been proven to be essential in human parasite diagnostics and understanding of their diversity. However, the lack of substantial data for comparative analysis is still a challenge in Trichuris trichiura research. To provide high quality mitogenomes, we utilized long-read sequencing technology of Oxford Nanopore Technologies (ONT) to better resolve repetitive regions and to construct de novo mitogenome assembly minimizing reference biases. In this study, we got three de novo assembled mitogenomes of T. trichiura isolated from Korean individuals. These circular complete mitogenomes of T. trichiura are 14,508 bp, 14,441 bp, and 14,440 bp in length. A total of 37 predicted genes were identified consisting of 13 protein-coding genes (PCGs), 22 transfer RNA (tRNAs) genes, two ribosomal RNA (rRNA) genes (rrnS and rrnL), and two non-coding regions. Interestingly, the assembled mitogenome has up to six times longer AT-rich regions than previous reference sequences, thus proving the advantage of long-read sequencing in resolving unreported non-coding regions. Furthermore, variant detection and phylogenetic analysis using concatenated protein coding genes, cox1, rrnL, and nd1 genes confirmed the distinct molecular identity of this newly assembled mitogenome while at the same time showing high genetic relationship with sequences from China or Tanzania. Our study provided a new set of reference mitogenome with better contiguity and resolved repetitive regions that could be used for meaningful phylogenetic analysis to further understand disease transmission and parasite biology.

Fig 1. Trichuris trichiura complete mitogenome circular map showing annotated features.

Schematic representation of the mitogenome including 13 Protein coding genes, 2 rRNAs and 22 tRNAs. AT-rich region that span 6× longer than reference mitogenomes lies between nd1 and nd2. Inner most ring shows the GC content and GC skew of each regions.

Fig 2. Heatmap of mitogenome variability.

Pairwise results of concatenated PCGs, cox1 region, and rrn L region of our assembled mitogenomes (TTK1-K3) and of Japan (TTJP), China (TTCN), and Uganda (TTUG) reference mitogenomes.

Fig 3. Genetic similarities between our assembled mitogenomes and the reference mitogenomes.

(A) Venn diagrams showing the unique and shared single nucleotide variations (SNVs) of TTK1 mitogenome in relation to each reference mitogenome. (B) Principal component analysis (PCA) using average nucleotide identity of each mitogenome.

Fig 4. Maximum likelihood tree showing the amount of genetic variability that has occurred between Trichuris species extracted from pigs, human and non-human primates.

Analysis is based on cox1 genes of our assembled sequence together with sequences reported and used by Cavallero et al., 2019 [37] and Doyle et al., 2022 [38]. A total of 106 sequences were used including the outgroup. Bootstrap values of branches with less than 85 value were not presented. Triangles represent collapsed cluster of group of taxa or clade within the tree.

Fig 5. Maximum likelihood tree showing the amount of genetic variability that has occurred between Trichuris species extracted from human and non-human primates.

Analysis is based on rrnL genes of our assembled sequence together with sequences reported and used by Cavallero et al., 2019 [37] and Doyle et al., 2022 [38]. A total of 98 sequences were used including the outgroup. Bootstrap values of branches with less than 60 value were not presented. Triangles represent collapsed cluster of group of taxa or clade within the tree.

Fig 6. Maximum likelihood tree showing the amount of genetic variability between Trichuris species extracted from human and non-human primates.

Analysis is based on nd1 genes of our assembled sequence together with sequences reported and used by Cavallero et al., 2019 [37] and Doyle et al., 2022 [38]. A total of 39 sequences were used including the outgroup. Bootstrap values of branches with less than 60 value were not presented. Triangles represent collapsed cluster of group of taxa or clade within the tree.