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

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.

SUBMITTER: Kinkar L

PROVIDER: S-EPMC7449408 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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

Kinkar Liina L Young Neil D ND Sohn Woon-Mok WM Stroehlein Andreas J AJ Korhonen Pasi K PK Gasser Robin B RB

PLoS neglected tropical diseases 20200826 8

<h4>Background</h4>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- ...[more]

PMID: 32845881

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Project description:BackgroundClonorchiasis, caused by the liver fluke Clonorchis sinensis, remains a serious public health issue in Asia, especially in China, and its relationship with cholangiocarcinoma has highlighted the importance of C. sinensis infection. Proteins containing tandem repeats (TRs) are found in a variety of parasites and, as targets of B-cell responses, are valuable for the serodiagnosis of parasite infections. Here, we identified a novel C. sinensis-specific antigen, Cs1, containing TRs, and investigated its diagnostic value, other immunological properties, and tissue distribution.Methodology/principal findingsA partial Cs1 cDNA sequence was cloned by screening an adult C. sinensis cDNA expression library. The full-length Cs1 cDNA was obtained by 5' rapid amplification of cDNA ends. The deduced Cs1 protein consists of a signal peptide and five TRs of 21 amino acids. The recombinant Cs1 (rCs1) was constructed and purified. rCs1 showed higher sensitivity (94.3%) and specificity (94.4%) than the C. sinensis excretory-secretory products (ESPs) according to ELISA of 114 serum samples. Native Cs1 was identified in C. sinensis ESPs and crude antigens of adult C. sinensis by western blotting using an anti-rCs1 monoclonal antibody. ELISA of recombinant peptides of different Cs1 regions demonstrated that the TR region was immunodominant in Cs1. Immunohistochemistry and confocal microscopy revealed that Cs1 is located in a granule-like structure surrounding the acetabulum of C. sinensis adults that has not previously been described.Conclusions/significanceWe identified a novel C. sinensis-specific TR protein, Cs1, which is an antigen of high serological significance, compared with C. sinensis ESPs. The deduced features of Cs1 show a unique structure containing TRs and a signal peptide and the TR region is immunodominant in Cs1. This provides a basis for targeted screens of other antigens. The novel structure in which Cs1 is located also deserves further investigation.

Project description:BackgroundDe novo genome assembly is essential to modern genomics studies. As it is not biased by a reference, it is also a useful method for studying genomes with high variation, such as cancer genomes. De novo short-read assemblers commonly use de Bruijn graphs, where nodes are sequences of equal length k, also known as k-mers. Edges in this graph are established between nodes that overlap by [Formula: see text] bases, and nodes along unambiguous walks in the graph are subsequently merged. The selection of k is influenced by multiple factors, and optimizing this value results in a trade-off between graph connectivity and sequence contiguity. Ideally, multiple k sizes should be used, so lower values can provide good connectivity in lesser covered regions and higher values can increase contiguity in well-covered regions. However, current approaches that use multiple k values do not address the scalability issues inherent to the assembly of large genomes.ResultsHere we present RResolver, a scalable algorithm that takes a short-read de Bruijn graph assembly with a starting k as input and uses a k value closer to that of the read length to resolve repeats. RResolver builds a Bloom filter of sequencing reads which is used to evaluate the assembly graph path support at branching points and removes paths with insufficient support. RResolver runs efficiently, taking only 26 min on average for an ABySS human assembly with 48 threads and 60 GiB memory. Across all experiments, compared to a baseline assembly, RResolver improves scaffold contiguity (NGA50) by up to 15% and reduces misassemblies by up to 12%.ConclusionsRResolver adds a missing component to scalable de Bruijn graph genome assembly. By improving the initial and fundamental graph traversal outcome, all downstream ABySS algorithms greatly benefit by working with a more accurate and less complex representation of the genome. The RResolver code is integrated into ABySS and is available at https://github.com/bcgsc/abyss/tree/master/RResolver .

Project description:BackgroundEchinococcus tapeworms cause a severe helminthic zoonosis called echinococcosis. The genus comprises various species and genotypes, of which E. granulosus (sensu stricto) represents a significant global public health and socioeconomic burden. Mitochondrial (mt) genomes have provided useful genetic markers to explore the nature and extent of genetic diversity within Echinococcus and have underpinned phylogenetic and population structure analyses of this genus. Our recent work indicated a sequence gap (> 1 kb) in the mt genomes of E. granulosus genotype G1, which could not be determined by PCR-based Sanger sequencing. The aim of the present study was to define the complete mt genome, irrespective of structural complexities, using a long-read sequencing method.MethodsWe extracted high molecular weight genomic DNA from protoscoleces from a single cyst of E. granulosus genotype G1 from a sheep from Australia using a conventional method and sequenced it using PacBio Sequel (long-read) technology, complemented by BGISEQ-500 short-read sequencing. Sequence data obtained were assembled using a recently-developed workflow.ResultsWe assembled a complete mt genome sequence of 17,675 bp, which is > 4 kb larger than the complete mt genomes known for E. granulosus genotype G1. This assembly includes a previously-elusive tandem repeat region, which is 4417 bp long and consists of ten near-identical 441-445 bp repeat units, each harbouring a 184 bp non-coding region and adjacent regions. We also identified a short non-coding region of 183 bp, which includes an inverted repeat.ConclusionsWe report what we consider to be the first complete mt genome of E. granulosus genotype G1 and characterise all repeat regions in this genome. The numbers, sizes, sequences and functions of tandem repeat regions remain to be studied in different isolates of genotype G1 and in other genotypes and species. The discovery of such 'new' repeat elements in the mt genome of genotype G1 by PacBio sequencing raises a question about the completeness of some published genomes of taeniid cestodes assembled from conventional or short-read sequence datasets. This study shows that long-read sequencing readily overcomes the challenges of assembling repeat elements to achieve improved genomes.

Dataset Information

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

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

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