A high-quality de novo genome assembly based on nanopore sequencing of a wild-caught coconut rhinoceros beetle (Oryctes rhinoceros)

Abstract

Background An optimal starting point for relating genome function to organismal biology is a high-quality nuclear genome assembly, and long-read sequencing is revolutionizing the production of this genomic resource in insects. Despite this, nuclear genome assemblies have been under-represented for agricultural insect pests, particularly from the order Coleoptera. Here we present a de novo genome assembly and structural annotation for the coconut rhinoceros beetle, Oryctes rhinoceros (Coleoptera: Scarabaeidae), based on Oxford Nanopore Technologies (ONT) long-read data generated from a wild-caught female, as well as the assembly process that also led to the recovery of the complete circular genome assemblies of the beetle’s mitochondrial genome and that of the biocontrol agent, Oryctes rhinoceros nudivirus (OrNV). As an invasive pest of palm trees, O. rhinoceros is undergoing an expansion in its range across the Pacific Islands, requiring new approaches to management that may include strategies facilitated by genome assembly and annotation. Results High-quality DNA isolated from an adult female was used to create four ONT libraries that were sequenced using four MinION flow cells, producing a total of 27.2 Gb of high-quality long-read sequences. We employed an iterative assembly process and polishing with one lane of high-accuracy Illumina reads, obtaining a final size of the assembly of 377.36 Mb that had high contiguity (fragment N50 length = 12 Mb) and accuracy, as evidenced by the exceptionally high completeness of the benchmarked set of conserved single-copy orthologous genes (BUSCO completeness = 99.1%). These quality metrics place our assembly ahead of the published Coleopteran genomes, including that of an insect model, the red flour beetle (Tribolium castaneum). The structural annotation of the nuclear genome assembly contained a highly-accurate set of 16,371 protein-coding genes, with only 2.8% missing BUSCOs, and the expected number of non-coding RNAs. The number and structure of paralogous genes in a gene family like Sigma GST is lower than in another scarab beetle (Onthophagus taurus), but higher than in the red flour beetle (Tribolium castaneum), which suggests expansion of this GST class in Scarabaeidae. The quality of our gene models was also confirmed with the correct placement of O. rhinoceros among other members of the rhinoceros beetles (subfamily Dynastinae) in a phylogeny based on the sequences of 95 protein-coding genes in 373 beetle species from all major lineages of Coleoptera. Finally, we provide a list of 30 candidate dsRNA targets whose orthologs have been experimentally validated as highly effective targets for RNAi-based control of several beetles. Conclusions The genomic resources produced in this study form a foundation for further functional genetic research and management programs that may inform the control and surveillance of O. rhinoceros populations, and we demonstrate the efficacy of de novo genome assembly using long-read ONT data from a single field-caught insect.

Omics in the Red Palm Weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae): A Bridge to the Pest

“…Developments in library preparation and assembly software also allow projects to deal more efficiently with heterozygosity so that the laborious process of creating inbred strains is no longer necessary for genome sequencing. Research efforts have capitalized on these advancements to generate high-quality genomic resources for multiple beetles, including major agricultural pests [ 45 , 46 , 47 , 48 , 49 , 50 , 51 ].…”

The Genome of the Soybean Gall Midge (Resseliella maxima)

“…However, long-read sequencing technologies (e.g., Pacific Biosciences (PacBio) and Oxford Nanopore) are contributing to improvement in quality of genome assembly, producing assemblies ~48X more contiguous than short-read based approaches (Hotaling et al, 2021; Li et al, 2019). Other pest insect genomes have recently been sequenced using nanopore-only approaches such as the black carpenter ant ( Camponotus pennsylvanicus (De Geer), Hymenoptera: Formicidae) and the coconut rhinoceros beetle ( Oryctes rhinoceros [L.] (Coleoptera: Scarabaeidae)) (Faulk, 2022; Filipović et al, 2022). Here we not only assembled a high-quality genome from long reads, but were able to do so from pooled samples.…”

MtGenome for the cost of a gene: Long-amplicon PacBio and Nanopore sequencing of hundreds of full mitochondrial genomes

“…If so, it will open-up cost-effective mtGenome sequencing in field settings and areas with limited laboratory resources [5]. One can expect to recover 4-20 Gb of data on a single MinIon flowcell [43][44][45][46] which is much higher than the 1.6 Gb we recovered here on the fresh MinIon flowcell, and generally amplicons are expected to yield higher throughput than genomic DNA. We may have recovered higher throughput on our ONT sequencing if we had prepared more input library for sequencing and used it entirely on the fresh flowcell rather than splitting it between the two as we ran out of library even though we still had active pores.…”