Genome Assembly Research Articles

Transposable element accumulation drives genome size increase in Hylesia metabus (Lepidoptera: Saturniidae), an urticating moth species from South America

Abstract We present the first nuclear genome assembly and a complete mitogenome for Hylesia metabus (Arthropoda; Insecta; Lepidoptera; Saturniidae). The assembled nuclear genome sequence is 1,271 Mb long, which is among the 10 largest lepidopteran genome assemblies published to date. It is scaffolded in 31 pseudo chromosomes, has a BUSCO score of 99.5%, and has a highly conserved synteny compared to phylogenetically close species. Repetitive elements make up 67% of the nuclear genome and are mainly located in intergenic regions, among which LINEs were predominant, with CR1-Zenon being the most abundant. Phylogenetic and comparative analyses of H. metabus assembly and 17 additional Saturniidae and Sphingidae assemblies suggested that an accumulation of repetitive elements likely led to the increased size of H. metabus’ genome. Gene annotation using Helixer identified 26,122 transcripts. The Z scaffold was identified using both a synteny analysis and variations of coverage for two resequenced male and female H. metabus. The H. metabus nuclear genome and mitogenome assemblies can be found and browsed on the BIPAA website and constitute useful resources for future population and comparative genomics studies.

Genome sequences of recombinant African swine fever virus isolated from domestic pigs in Vietnam

ABSTRACT African swine fever is a highly contagious and devastating disease of pigs. Here, we present the draft genome assemblies of three Vietnamese African s wine fever virus (ASFV) isolates: rASF1/2-avac02, rASF1/2-avac03, and rASF1/2-avac07. All isolates were closely related to the Chinese recombinant ASFV strains of genotypes I and II.

The T2T Genome of the Domesticated Silkworm Bombyx mori

Genome sequences contain the fundamental genetic information that largely determines the biology of a species. Over the past 20 years, advancements in high-throughput sequencing technologies and bioinformatics tools have matured, facilitating genome assembly and ushering in the telomere-to-telomere (T2T) era. Bombyx mori is renowned as a silk-producing insect and serves as an important model organism extensively studied across various fields of biology. In this study, we present the first assembled T2T genome by integrating HiFi, ultra-long ONT, NGS, and Hi-C data. This assembly comprises 450,267,439 base pairs from 28 chromosomes and includes annotations for a total of 18,253 protein-coding genes. A completeness evaluation revealed that 99.1% of conserved single-copy genes were included, as determined by a BUSCO analysis. Furthermore, the consensus quality (QV) assessed through Merqury was recorded at 59.88. The proportion of repeat sequence achieved 60.77%, marking it as the highest reported value for B. mori to date. In comparison to previously published genomes, our assembly offers a more complete and higher quality representation, particularly concerning highly homologous tandem regions such as telomeres, rDNA clusters, and Gr family regions. Furthermore, our extensive experience in genome assembly, including sample preparation experience and assembly strategies to reduce complexity, will provide valuable references for other species aiming to achieve their own T2T genome assemblies.

Repeat-rich regions cause false positive detection of NUMTs: a case study in amphibians using an improved cane toad reference genome.

Mitochondrial DNA (mtDNA) has been widely used in genetics research for decades. Contamination from nuclear DNA of mitochondrial origin (NUMT) can confound studies of phylogenetic relationships and mtDNA heteroplasmy. Homology searches with mtDNA are widely used to detect NUMTs in the nuclear genome. Nevertheless, false positive detection of NUMTs is common when handling repeat-rich sequences, whilst fragmented genomes might result in missing true NUMTs. In this study, we investigated different NUMT detection methods and how the quality of the genome assembly affects them. We presented an improved nuclear genome assembly (aRhiMar1.3) of the invasive cane toad (Rhinella marina) with additional long-read Nanopore and 10x linked-read sequencing. The final assembly was 3.47 Gb in length with 91.3% of tetrapod universal single-copy orthologs (n=5,310), indicating the gene-containing regions were well assembled. We used three complementary methods (NUMTFinder, dinumt and PALMER) to study the NUMT landscape of the cane toad genome. All three methods yielded consistent results, showing very few NUMTs in the cane toad genome. Furthermore, we expanded NUMT detection analyses to other amphibians and confirmed a weak relationship between genome size and the number of NUMTs present in the nuclear genome. Amphibians are repeat-rich, and we show that the number of NUMTs found in highly repetitive genomes is prone to inflation when using homology-based detection without filters. Together, this study provides an exemplar of how to robustly identify NUMTs in complex genomes when confounding effects on mtDNA analyses are a concern.

Identification of RppSLN from an Elite Landrace: A Major Locus Conferring Resistance to Southern Corn Rust in Maize (Zea mays L.)

Southern corn rust (SCR) is one of the most destructive foliar diseases in maize (Zea mays L.), resulting in significant yield losses. Therefore, the continuous identification of disease-resistant germplasm and the deployment of resistant hybrids is essential for durably controlling SCR. The objective of this research was to identify and characterize resistance loci against SCR in maize to expand disease management strategies. Here, we identified a maize landrace with high resistance to SCR ‘Silunuo’ (SLN) approaching complete immunity. We backcrossed it with a susceptible inbred line, N531, to generate a stable SCR-resistant introgression line N531_R. By crossing it with F35 (a susceptible inbred line), we created a large F2 segregating population and mapped a major SCR-resistant locus on chromosome 10, known as RppSLN. Based on the genome assembly and annotation, we found that RppSLN harbors two NBS-LRR (nucleotide binding site–leucine-rich repeat) genes, namely Zmays10G000430 and Zmays10G000440. These NBS-LRR genes were significantly induced during artificial inoculation with Puccinia polysora, suggesting that they might be candidate genes collectively contributing to the resistance level at this locus. In conclusion, this study identified a major SCR resistance locus directly isolated from a landrace, providing valuable support and information for expanding new disease-resistant germplasms and promoting the utilization of landraces.

High-quality genome assembly and annotation of the crested gecko (Correlophus ciliatus).

Correlophus ciliatus, or the crested gecko, is widely kept as a pet in many countries around the world due to its ease to care and bred and its high survival rate. However, there is limited number of genomic studies on the crested gecko. In this study, we generated a high-quality chromosome-level genome assembly of the crested gecko by combining Nanopore, Illumina, and Hi-C data. The genome assemble has a size of 1.66 Gb, with scaffold N50 of 109.97 Mb, and 99.52% of the scaffold anchored on 19 chromosomes. The BUSCO analysis indicated a gene completeness of 90.3% (n=7,480), including 6,673 (89.2%) single-copy genes and 84 (1.1%) duplicated genes. Additionally, we identified 21,065 protein-coding genes using the MAKER3 annotation toolkit, with 41.98% (697.51 Mb) consisting of repetitive elements. Among these, 21,037 genes were validated through InterProScan5. Our study is the first to report a chromosome-level genome for the crested gecko. It provides valuable genomic resources for understanding molecular mechanisms under many interesting traits of the species.

MosAIC: An annotated collection of mosquito-associated bacteria with high-quality genome assemblies.

Mosquitoes transmit medically important human pathogens, including viruses like dengue virus and parasites such as Plasmodium spp., the causative agent of malaria. Mosquito microbiomes are critically important for the ability of mosquitoes to transmit disease-causing agents. However, while large collections of bacterial isolates and genomic data exist for vertebrate microbiomes, the vast majority of work in mosquitoes to date is based on 16S rRNA gene amplicon data that provides limited taxonomic resolution and no functional information. To address this gap and facilitate future studies using experimental microbiome manipulations, we generated a bacterial Mosquito-Associated Isolate Collection (MosAIC) consisting of 392 bacterial isolates with extensive metadata and high-quality draft genome assemblies that are publicly available, both isolates and sequence data, for use by the scientific community. MosAIC encompasses 142 species spanning 29 bacterial families, with members of the Enterobacteriaceae comprising 40% of the collection. Phylogenomic analysis of 3 genera, Enterobacter, Serratia, and Elizabethkingia, reveal lineages of mosquito-associated bacteria isolated from different mosquito species in multiple laboratories. Investigation into species' pangenomes further reveals clusters of genes specific to these lineages, which are of interest for future work to test for functions connected to mosquito host association. Altogether, we describe the generation of a physical collection of mosquito-associated bacterial isolates, their genomic data, and analyses of selected groups in context of genome data from closely related isolates, providing a unique, highly valuable resource for research on bacterial colonisation and adaptation within mosquito hosts. Future efforts will expand the collection to include broader geographic and host species representation, especially from individuals collected from field populations, as well as other mosquito-associated microbes, including fungi, archaea, and protozoa.

Niche-Aware Metagenomic Screening for Enzyme Methioninase Illuminates Its Contribution to Metabolic Syntrophy.

The single-step methioninase-mediated degradation of methionine (as a sulfur containing amino acid) is a reaction at the interface of carbon, nitrogen, sulfur, and methane metabolism in microbes. This enzyme also has therapeutic application due to its role in starving auxotrophic cancer cells. Applying our refined in silico screening pipeline on 33,469 publicly available genome assemblies and 1878 metagenome assembled genomes/single-cell amplified genomes from brackish waters of the Caspian Sea and the Fennoscandian Shield deep groundwater resulted in recovering 1845 methioninases. The majority of recovered methioninases belong to representatives of phyla Proteobacteria (50%), Firmicutes (29%), and Firmicutes_A (13%). Prevalence of methioninase among anaerobic microbes and in the anoxic deep groundwater together with the relevance of its products for energy conservation in anaerobic metabolism highlights such environments as desirable targets for screening novel methioninases and resolving its contribution to microbial metabolism and interactions. Among archaea, majority of detected methioninases are from representatives of Methanosarcina that are able to use methanethiol, the sulfur containing product from methionine degradation, as a precursor for methanogenesis. Branching just outside these archaeal methioninases in the phylogenetic tree, we recovered three methioninases belonging to representatives of Patescibacteria reconstructed from deep groundwater metagenomes. We hypothesize that methioninase in Patescibacteria could contribute to their syntrophic interactions where their methanogenic partners/hosts benefit from the produced 2-oxobutyrate and methanethiol. Our results underscore the significance of accounting for specific ecological niche in screening for enzyme variates with desired characteristics. Finally, complementing of our findings with experimental validation of methioninase activity confirms the potential of our in silico screening in clarifying the peculiar ecological role of methioninase in anoxic environments.

Chromosome-level genome assembly of the smallscale yellowfin (Plagiognathops microlepis).

The small-scale yellowfin (Plagiognathops microlepis) is a highly valued species in East Asian aquaculture due to its adaptability and high yield. However, the lack of genomic data has impeded genetic research and breeding efforts. In this study, we utilize PacBio Hifi long-read sequencing and Hi-C technologies to construct a highly detailed genome of P. microlepis at the chromosomal level. The assembly encompasses 976.41 Mb, with an exceptional 99.84% distribution across 24 chromosomes. Notably, the contig N50 was 34.41 Mb and scaffold N50 was 38.38 Mb. The completeness of the P. microlepis genome assembly is underscored by a BUSCO score of 98.08%. A total of 25,389 protein-coding genes were identified, with a BUSCO score of 96.98%, and 99.85% of these genes were functionally annotated. Synteny relationships at the chromosome level with Danio rerio and Chanodichthys erythropterus genomes uncover small-scale chromosomal rearrangements. This high-fidelity genome assembly serves as a pivotal resource for forthcoming endeavors such as the genome structure, functional elements, comparative genomics, and evolutionary characteristics of P. microlepis and its relative species.

Novel toxin biosynthetic gene cluster in harmful algal bloom-causing Heteroscytonema crispum: Insights into the origins of paralytic shellfish toxins.

Caused by both eukaryotic dinoflagellates and prokaryotic cyanobacteria, harmful algal blooms (HABs) are events of severe ecological, economic, and public health consequence, and their incidence has become more common of late. Despite coordinated research efforts to identify and characterize the genomes of HAB-causing organisms, the genomic basis and evolutionary origins of paralytic shellfish toxins (PSTs) produced by HABs remain at best incomplete. The PST saxitoxin has an especially complex genomic architecture and enigmatic phylogenetic distribution, spanning dinoflagellates and multiple cyanobacterial genera. Using filtration and extraction techniques to target the desired cyanobacteria from non-axenic culture, coupled with a combination of short and long read sequencing, we generated a reference-quality hybrid genome assembly for Heteroscytonema crispum UTEX LB 1556, a freshwater, PST-producing cyanobacterium thought to have the largest known genome in its phylum. We report a complete, novel biosynthetic gene cluster for the PST saxitoxin. Leveraging this biosynthetic gene cluster, we find support for the hypothesis that PST production has appeared in divergent Cyanobacteria lineages through widespread and repeated horizontal gene transfer. This work demonstrates the utility of long-read sequencing and metagenomic assembly toward advancing our understanding of PST biosynthetic gene cluster diversity and suggests a mechanism for the origin of PST biosynthetic genes.

De novo whole-genome assembly and annotation of Coffea arabica var. Geisha, a high-quality coffee variety from the primary origin of coffee.

Geisha coffee is recognized for its unique aromas and flavors and accordingly, has achieved the highest prices in the specialty coffee markets. We report the development of a chromosome-level, well-annotated, genome assembly of Coffea arabica var. Geisha. Geisha is considered an Ethiopian landrace that represents germplasm from the Ethiopian center of origin of coffee. We used a hybrid de novo assembly approach combining two long-reads single molecule sequencing technologies, Oxford Nanopore and Pacific Biosciences, together with scaffolding with Hi-C libraries. The final assembly is 1.03GB in size with BUSCO assessment of the assembly completeness of 97.7% of single-copy orthologs clusters. RNAseq and IsoSeq data were used as transcriptional experimental evidence for annotation and gene prediction revealing the presence of 47,062 gene loci encompassing 53,273 protein-coding transcripts. Comparison of the assembly to the progenitor subgenomes separated the set of chromosome sequences inherited from C. canephora from those of C. eugenioides. Corresponding orthologs between the two Arabica varieties, Geisha and Red Bourbon, had a 99.67% median identity, higher than what we observe with the progenitor assemblies (median 97.28%). Both Geisha and Red Bourbon contain a recombination event on Chromosome 10 relative to the two progenitors that must have happened before the geographical separation of the two varieties, consistent with a single allopolyploidization event giving rise to C. arabica. Broadening the availability of high-quality genome assemblies of Coffea arabica varieties, paves the way for understanding the evolution and domestication of coffee, as well as the genetic basis and environmental interactions of why a variety like Geisha is capable of producing beans with such exceptional and unique high-quality.

The Genomic SSR Millets Database (GSMDB): enhancing genetic resources for sustainable agriculture.

The global population surge demands increased food production and nutrient-rich options to combat rising food insecurity. Climate-resilient crops are vital, with millets emerging as superfoods due to nutritional richness and stress tolerance. Given limited genomic information, a comprehensive genetic resource is crucial to advance millet research. Whole-genome sequencing provides an unprecedented opportunity, and molecular genetic methodologies, particularly simple sequence repeats (SSRs), play a pivotal role in DNA fingerprinting, constructing linkage maps, and conducting population genetic studies. SSRs are composed of repetitive DNA sequences where one to six nucleotides are repeated in tandem and distributed throughout the genome. Different millet species exhibit genomic variations attributed to the presence of SSRs. While SSRs have been identified in a few millet species, the existing information only covers some of the sequenced genomes. Moreover, there is an absence of complete gene annotation and visualization features for SSRs. Addressing this disparity and leveraging the de-novo millet genome assembly available from the NCBI, we have developed the Genomic SSR Millets Database (GSMDB; https://bioinfo.icgeb.res.in/gsmdb/). This open-access repository provides a web-based tool offering search functionalities and comprehensive details on 6.747645 million SSRs mined from the genomic sequences of seven millet species. The database, featuring unrestricted public access and JBrowse visualization, is a pioneering resource for the research community dedicated to advancing millet cultivars and related species. GSMDB holds immense potential to support myriad studies, including genetic diversity assessments, genetic mapping, marker-assisted selection, and comparative population investigations aiming to facilitate the millet breeding programs geared toward ensuring global food security. Database URL: https://bioinfo.icgeb.res.in/gsmdb/.

Benchmarking of Hi-C tools for scaffolding plant genomes obtained from PacBio HiFi and ONT reads

The implementation of Hi-C reads in the de novo genome assembly process allows the ordering of large regions of the genome in scaffolds and the generation of chromosome-level assemblies. Several bioinformatics tools have been developed for genome scaffolding with Hi-C, and each tool has advantages and disadvantages that need to be carefully evaluated before their adoption. We generated two de novo assemblies of Arabidopsis thaliana obtained from the same raw PacBio HiFi and Oxford Nanopore Technologies data. We scaffolded the assemblies implementing Hi-C reads with the scaffolders 3D-DNA, SALSA2, and YaHS, with the aim of identifying the tool providing the most accurate assembly. The scaffolded assemblies were evaluated according to contiguity, completeness, accuracy, and structural correctness. In our analysis, YaHS proved to be the best-performing bioinformatics tool for scaffolding de novo genome assemblies in Arabidopsis thaliana.

Transcriptional dynamics and tissue-specific expression patterns of transposable elements in orthopteran insects.

Transposable elements (TEs) are prevalent in the genomes of orthopteran insects, contributing significantly to their genome evolution and diversity. In light of the existing gap in our understanding of TEs transcript dynamics in orthopteran insects, we recognize the critical need to undertake comprehensive analyses in this area. Therefore, we have decided to delve into the characterization of TE transcripts, their abundance profiles, and the formation of chimeric transcripts using RNA-seq data and genome assemblies. The transcript analysis of TEs across various species revealed significant differences in TE abundance and expression patterns. In particular, Schistocerca americana exhibited twice the number of transcripts within the genus Schistocerca compared to the average of other species, while Gryllus bimaculatus displayed the lowest number of transcripts. Despite this, all Schistocerca species shared similar fractions of TE transcripts at the clade level, with DNA transposons (45%) being the most abundant, followed by LINE (19%) and LTR elements (18%). Interestingly, Acrida cinerea displayed different TE abundance patterns compared to Schistocerca species, particularly with an increased proportion of LTR transcripts, accounting for 31% of the total transcripts. Further analysis revealed tissue-specific transcriptional activity of TE clades, with notable differences between male and female specimens. In Gryllus bimaculatus, TEs were highly transcribed across ovaries and gut tissues in females compared to male testes and gut. Conversely, Gastrimargus marmoratus displayed higher TE transcription in male tissues compared to females, indicating species-specific expression patterns. A similar pattern has been observed in Acrida cinerea, except in female gonads, where 4618 TEs were transcribed compared to 3757 in male gonads. Despite these variations, no correlation was found between genome size and TE transcript abundance. Additionally, highly conserved TEs were involved in the formation of chimeric transcripts, indicating potential regulatory roles in gene expression. The expression quantification analysis of chimeric TEs and genes revealed tissue-specific expression patterns, and TEs do not control the overall expression of all genes except some, suggesting regulatory roles of TEs in gene expression. Overall, our study underscores tissue-specific variations in TE expression and transcript abundance among different species. Additionally, our findings suggest the involvement of highly conserved TEs in the formation of chimeric transcripts across different species.

The genome sequence of bronze sap hoverfly, Ferdinandea cuprea (Scopoli, 1763)

We present a genome assembly from an individual Ferdinandea cuprea (bronze sap hoverfly; Arthropoda; Insecta; Diptera; Syrphidae). The genome sequence has a total length of 560.20 megabases. Most of the assembly (99.92%) is scaffolded into 5 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 16.0 kilobases in length. Gene annotation of this assembly on Ensembl identified 12,662 protein-coding genes.

The complete genome assembly of Nicotiana benthamiana reveals the genetic and epigenetic landscape of centromeres.

Nicotiana benthamiana is a model organism widely adopted in plant biology. Its complete assembly remains unavailable despite several recent improvements. To further improve its usefulness, we generate and phase the complete 2.85 Gb genome assembly of allotetraploid N. benthamiana. We find that although Solanaceae centromeres are widely dominated by Ty3/Gypsy retrotransposons, satellite-based centromeres are surprisingly common in N. benthamiana, with 11 of 19 centromeres featured by megabase-scale satellite arrays. Interestingly, the satellite-enriched and satellite-free centromeres are extensively invaded by distinct Gypsy retrotransposons which CENH3 protein more preferentially occupies, suggestive of their crucial roles in centromere function. We demonstrate that ribosomal DNA is a major origin of centromeric satellites, and mitochondrial DNA could be employed as a core component of the centromere. Subgenome analysis indicates that the emergence of satellite arrays probably drives new centromere formation. Altogether, we propose that N. benthamiana centromeres evolved via neocentromere formation, satellite expansion, retrotransposon enrichment and mtDNA integration.

The chromosome-level genome assembly and annotation of an invasive forest pest Obolodiplosis robiniae.

Biological invasion is a major global problem, leading to the loss of biodiversity and species extinction, and causing huge economic losses to countries. Obolodiplosis robiniae is a major invasive forest pest that has caused economic losses in Asia and Europe. Here, the chromosome- level genome of O. robiniae was assembled using the PacBio platform and Hi-C technology. A contig-level genome with a length of 199.49 Mb and a contig N50 of 4.66 Mb was assembled. Approximately 98.05% of contigs were successfully anchored to four chromosomes using Hi-C assisted genome assembly. The genome integrity was assessed to be 90.3% based on BUSCOs analysis. The high-quality genome provides valuable data for the study of invasive species, and a foundation for the understanding the biology and ecology of O. robiniae.

Mechanical Behaviour Assessment and Optimization of Construction Sequence for an HSS Vierendeel Truss

Long-span Vierendeel trusses in construction are challenged by smooth force transfer and critical stress control concerns. In this article, construction sequences characterized by loading/unloading due to various assemblies of the branches and/or temporary lattice column supports are focused on to reproduce their related mechanical behaviours in finite element modelling. Typical bending moment and shear force diagrams for hollow structure section (HSS) chords are analysed first, and then combined with a corrected assessment index in the development of an optimized construction scheme through effective discussion. It is observed that the branch assembly sequence has significant but reversed influences on either the bending moments or the shear forces of the interior support and the midspan of the HSS chords. The influence of the branch assembly sequence on the bending moment and shear force are compromised with the introduction of a temporary support. The corrected assessment index superimposed by the internal force and the deflection is demonstrated properly to represent the strength and the serviceability requirements, especially for characterizing the removal of a temporary support after the completed construction of a twelve-panel Vierendeel truss for the roof of an assembly hall.

Chromosome-level genome assembly of Indo-Pacific king mackerel (Scomberomorus guttatus).

The Indo-Pacific king mackerel, scientifically known as Scomberomorus guttatus, is a valued marine species that holds significant commercial importance in the Indo-Pacific region. However, the lack of genomic resources has hindered a comprehensive understanding of this species. In this study, we constructed a genome of Indo-Pacific king mackerel at the chromosome level using a combination of PacBio HiFi reads and a chromosome contact map (Hi-C). The resulting genome had high contig and scaffold N50 values of 8.84 Mb and 32.9 Mb, respectively. In the genome assembly, which is 797.66 Mb in size and consists of 24 chromosomes, we also identified 35.89% repetitive elements and predicted 25,886 protein-coding genes. Our study not only benefits to reveal the possible mechanism of adaptive evolution in this fish, but also offers insight for the future sustainable management of these valuable biological resources.

A Whole-Genome Survey and the Mitochondrial Genome of Acanthocepola indica Provide Insights into Its Phylogenetic Relationships in Priacanthiformes

Acanthocepola indica, a deep-sea snake fish, is primarily found in the Indo-west Pacific region, including India, Korea, Japan, and the South China Sea. The taxonomic classification of A. indica based on morphological characteristics remains inaccurate and unclear. In this study, we utilized next-generation sequencing to generate comprehensive genomic data for A. indica. The estimated genome size of A. indica was 422.95 Mb, with a heterozygosity ratio of 1.02% and a sequence repeat ratio of 22.43%. Our analysis suggested that A. indica is diploid, and the draft genome assembly consists of 1,059,784 contigs with a contig N50 of 1942 bp. We identified a total of 444,728 simple sequence repeats in the genome of A. indica. Furthermore, we successfully assembled the complete mitochondrial genome (16,439 bp) of A. indica, which included 13 protein-coding genes, 22 tRNA genes and 2 rRNA genes. Phylogenetic analysis based on mitochondrial genomes revealed that A. indica is closely related to Acanthocepola krusensternii and Cepola schlegelii, providing evidence that the family Cepolidae belongs to the order Priacanthiformes. Population size dynamics analysis indicated that A. indica experienced a bottleneck effect during the Pleistocene Glacial Epoch, likely due to the changes in glacial cycles and sea level fluctuations since ~800 Kya.