Dataset Information

Nanopore Sequencing Resolves Elusive Long Tandem-Repeat Regions in Mitochondrial Genomes.

ABSTRACT: Long non-coding, tandem-repetitive regions in mitochondrial (mt) genomes of many metazoans have been notoriously difficult to characterise accurately using conventional sequencing methods. Here, we show how the use of a third-generation (long-read) sequencing and informatic approach can overcome this problem. We employed Oxford Nanopore technology to sequence genomic DNAs from a pool of adult worms of the carcinogenic parasite, Schistosoma haematobium, and used an informatic workflow to define the complete mt non-coding region(s). Using long-read data of high coverage, we defined six dominant mt genomes of 33.4 kb to 22.6 kb. Although no variation was detected in the order or lengths of the protein-coding genes, there was marked length (18.5 kb to 7.6 kb) and structural variation in the non-coding region, raising questions about the evolution and function of what might be a control region that regulates mt transcription and/or replication. The discovery here of the largest tandem-repetitive, non-coding region (18.5 kb) in a metazoan organism also raises a question about the completeness of some of the mt genomes of animals reported to date, and stimulates further explorations using a Nanopore-informatic workflow.

SUBMITTER: Kinkar L

PROVIDER: S-EPMC7918261 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Nanopore Sequencing Resolves Elusive Long Tandem-Repeat Regions in Mitochondrial Genomes.

Kinkar Liina L Gasser Robin B RB Webster Bonnie L BL Rollinson David D Littlewood D Timothy J DTJ Chang Bill C H BCH Stroehlein Andreas J AJ Korhonen Pasi K PK Young Neil D ND

International journal of molecular sciences 20210211 4

Long non-coding, tandem-repetitive regions in mitochondrial (mt) genomes of many metazoans have been notoriously difficult to characterise accurately using conventional sequencing methods. Here, we show how the use of a third-generation (long-read) sequencing and informatic approach can overcome this problem. We employed Oxford Nanopore technology to sequence genomic DNAs from a pool of adult worms of the carcinogenic parasite, <i>Schistosoma haematobium</i>, and used an informatic workflow to d ...[more]

PMID: 33670420

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Project description: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.

Dataset Information

Nanopore Sequencing Resolves Elusive Long Tandem-Repeat Regions in Mitochondrial Genomes.

Publications

Nanopore Sequencing Resolves Elusive Long Tandem-Repeat Regions in Mitochondrial Genomes.

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