First record of a tandem-repeat region within the mitochondrial genome of Clonorchis sinensis using a long-read sequencing approach

Abstract

Author summary In the present study, we characterised the complete mitochondrial genome ofClonorchis sinensis-a carcinogenic liver fluke. To do this, we sequenced from total genomic DNA from multiple adult worms using a new method (Oxford Nanopore technology) to obtain data for long stretches of DNA, and then assembled these data to construct a mitochondrial genome of 18,304 bp, containing a > 4.5 kb-long tandem-repetitive region-not previously detected in this species. The results demonstrate that this method is effective at sequencing long and complex non-coding elements-not achievable using conventional techniques. The discovery of this long tandem-repetitive region inC.sinensisprovides an opportunity to now explore its origin(s) and length/sequence diversity in populations of this species, and also to characterise its function(s). The technological approach employed here should have broad applicability to characterise previously-elusive non-coding mitochondrial genomic regions in a wide range of taxa. Background Mitochondrial genomes provide useful genetic markers for systematic and population genetic studies of parasitic helminths. Although many such genome sequences have been published and deposited in public databases, there is evidence that some of them are incomplete relating to an inability of conventional techniques to reliably sequence non-coding (repetitive) regions. In the present study, we characterise the complete mitochondrial genome-including the long, non-coding region-of the carcinogenic Chinese liver fluke,Clonorchis sinensis, using long-read sequencing. Methods The mitochondrial genome was sequenced from total high molecular-weight genomic DNA isolated from a pool of 100 adult worms ofC.sinensisusing the MinION sequencing platform (Oxford Nanopore Technologies), and assembled and annotated using an informatic approach. Results From > 93,500 long-reads, we assembled a 18,304 bp-mitochondrial genome forC.sinensis. Within this genome we identified a novel non-coding region of 4,549 bp containing six tandem-repetitive units of 719-809 bp each. Given that genomic DNA from pooled worms was used for sequencing, some variability in length/sequence in this tandem-repetitive region was detectable, reflecting population variation. Conclusions ForC.sinensis, we report the complete mitochondrial genome, which includes a long (> 4.5 kb) tandem-repetitive region. The discovery of this non-coding region using a nanopore-sequencing/informatic approach now paves the way to investigating the nature and extent of length/sequence variation in this region within and among individual worms, both within and amongC.sinensispopulations, and to exploring whether this region has a functional role in the regulation of replication and transcription, akin to the mitochondrial control region in mammals. Although applied toC.sinensis, the technological approach established here should be broadly applicable to characterise complex tandem-repetitive or homo-polymeric regions in the mitochondrial genomes of a wide range of taxa.