Long-read sequencing reveals a 4.4 kb tandem repeat region in the mitogenome of Echinococcus granulosus (sensu stricto) genotype G1

Abstract

Background Echinococcus 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. Methods We 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. Results We 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. Conclusions We 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.

Molecular phylogenetics and mitogenomics of three avian dicrocoeliids (Digenea: Dicrocoeliidae) and comparison with mammalian dicrocoeliids

“…Similar regions in other studies of mt genomes have also been shown as problematic [46,47]. Therefore, the PacBio single-molecule real-time sequencing method has been used by researchers in recent studies to characterize long and difficult to sequence repetitive regions of flatworm mitogenomes [48,49]. Nonetheless, the coding regions of the mt genomes of Brachylecithum sp.…”

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

“…All 12 protein-encoding genes, 22 tRNAs and two rRNAs had high sequence similarities (> 99.2%) to those in published mitochondrial genomes and occurred in the same order. However, there is clear evidence [17,18,38] that conventional sequencing methods are not suited to the sequencing of long non-coding regions in mitochondrial genomes. This obstacle has been overcome through the use of nanopore-sequencing, which bodes well for future mitochondrial genome investigations.…”

“…With the advent of 'short-read' sequencing (e.g., Illumina) came the confidence that sequencing at high coverage in a high throughput manner would readily allow the sequencing and assembly of complete mitochondrial genomes, because of their relatively small size (~14 kb ± 1 kb in flatworms; [14]). However, there have been challenges with sequencing through tandem-repetitive elements and regions with a biased nucleotide composition using Sanger and short-read technologies [16][17][18], and little attention has been paid to the impact of these issues.…”

“…Despite our efforts using a PCRbased sequencing strategy, we were not able to sequence this gap. However, employing a single molecule, real-time (SMRT) sequencing technology, we obtained long sequence reads that bridged the entire gap, allowing us to characterise a 4,417 bp-long tandem-repetitive region consisting of ten near-identical repeat units (441-445 bp), each harbouring a 184 bp non-coding region and flanking regions [17]. Although three mitochondrial genomes for E. granulosus genotype G1 had been published and/or deposited in public gene databases (including Gen-Bank), closing this gap allowed us to define (what we considered to be) the first complete mt genome (17,675 bp) for this genotype, being > 4 kb larger than any previously reported genome for this taxon.…”

Comprehensive characterization of plasma cell-free Echinococcus spp. DNA in echinococcosis patients using ultra-high-throughput sequencing

“…DNA features exploration. Since a more complete mitochondrial genome of E. granulosus has been published[45], we replaced the mitochondrial sequence of Tsai, et al[44] with the most updated one. Read pairs from E. granulosus annotated samples and E. multilocularis annotated samples were pooled separately and remapped with BWA (v0.7.16)[46] to their corresponding references to get the mapping positions and fragment length.…”