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

Liina Kinkar; Neil D Young; Woon-Mok Sohn; Andreas J Stroehlein; Pasi K Korhonen; Robin B Gasser

doi:10.1371/journal.pntd.0008552

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

PLoS Negl Trop Dis. 2020 Aug 26;14(8):e0008552. doi: 10.1371/journal.pntd.0008552. eCollection 2020 Aug.

Authors

Liina Kinkar¹, Neil D Young¹, Woon-Mok Sohn², Andreas J Stroehlein¹, Pasi K Korhonen¹, Robin B Gasser¹

Affiliations

¹ Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, Australia.
² Department of Parasitology and Tropical Medicine, and Institute of Health Sciences, Gyeongsang National University College of Medicine, Jinju, Korea.

Abstract

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 of C. sinensis using 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 for C. 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: For C. 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 among C. sinensis populations, 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 to C. 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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Base Sequence
Clonorchis sinensis / genetics*
DNA / isolation & purification
Genome, Mitochondrial*
Tandem Repeat Sequences / genetics*

Substances

DNA

Grants and funding

Project support was through Australian Research Council (ARC) grants LP180101334 (N.D.Y. and P.K.K.) and LP180101085 (R.B.G.) and Yourgene Health Singapore. N.D.Y. and P.K.K. were recipients of Career Development and Early Career Research Fellowships, respectively, from the National Health and Medical Research Council (NHMRC) of Australia. The LIEF HPC-GPGPU Facility is supported by ARC LIEF Grant LE170100200. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.