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bioRxiv2026

Modular Assembly of an Infectious Clone of the Akata strain of EBV using Synthetic Genomics Methods in Yeast

We have used synthetic biology recombination methods in yeast to build herpes simplex virus type-1 (HSV-1) and human cytomegalovirus (HCMV) genomes from multiple fragments. The genomes were built using transformation-associated recombination (TAR) in yeast, by virtue of overlapping sequences between the different fragments. This study demonstrates the successful assembly of the Epstein-Barr virus (EBV) genome. We used as the model genome, the Akata Burkitts lymphoma genome, specifically the BX1 genome which encodes a neomycin selectable marker and a GFP expression cassette in the BXLF1 region. The 171.3 kb genome was first deconstructed into 11 fragments in silico, each having 80 bp overlapping sequence between the fragments. The 11 fragments (TAR 1 to TAR 11) were cloned using TAR in yeast, analyzed by restriction enzyme analyses and Nanopore sequencing to validate the cloned fragment. The EBV genome was built in two stages: TAR fragments 1 to 6 and TAR fragments 7 to 11 were assembled to generate two half-genomes. The whole genome (TAR 1-11) was then assembled by joining TAR 1-6 with TAR 7-11. Complete EBV genomes were examined by PCR assays and restriction enzyme analyses and then transfected into HEK-293 cells to generate virus producer cell lines. The HEK-293 cell clones were tested for virus production following lytic induction using baculovirus transduction of Zta, Rta and glycoprotein B (BALF4). The supernatants from these induced cells were harvested and used to infect Raji cells. This analysis revealed a significant number of cells displaying strong GFP fluorescence indicative of infectious virus. We used this supernatant virus to infect primary B cells and were able to derive lymphoblastoid cell lines (LCL) indicative of the ability of this virus to transform B cells. We tested this method for engineering different mutations. Two mutations were made, one in Zta and the other in the small capsid protein (BFRF3). Mutations were engineered in the TAR plasmid in which the genes reside and after sequence validation, assembled into the TAR 1-6 half genome and then the TAR 1-11 genome, which was used to generate HEK-293 cell clones. For the {Delta}Zta cell lines, we could detect virus in the supernatants only if baculovirus expressing Zta in trans was included, this {Delta}Zta EBV virus could transform B cells. The small capsid protein (BFRF3) decorates the capsid shell and is required for capsid assembly in a self-assembly system. When the HEK-293 cell clones were induced using co-expression of Zta, Rta and gB, no virus was detected in the culture supernatants. However, if we provided BFRF3 in trans using baculovirus expressing this protein, virus was detected in the supernatants. This provides the first report of the essential role of the small capsid protein in EBV-infected cells.

Synthetic Biology↗

bioRxiv2026

An introgressed haplotype decouples phenotype from genome-wide ancestry

Introgression redistributes genetic variation among diverging lineages, shaping evolution-ary trajectories and contributing to phenotypic evolution. Yet how localized introgressed genomic regions persist despite extensive genomic homogenization remains poorly under-stood. Here, we investigate the evolutionary history of the northeastern Iranian great tit (Parus major intermedius), a grey-plumaged member of the great tit complex occurring at the eastern range margin of the green- and yellow-plumaged major lineage, adjacent to the grey-plumaged Central Asian bokharensis lineage, and long regarded as a putative hybrid. We find that P. m. intermedius retains predominantly major-derived genomic ancestry despite its grey plumage, revealing extensive genomic homogenization across the genome. Surprisingly, a single localized introgressed haplotype on chromosome 24 retains bokharensis-derived ancestry, exhibits elevated genomic differentiation relative to the genomic background, and overlaps the carotenoid-processing gene BCO2, a strong candidate underly-ing plumage pigmentation. Our findings provide a genomic explanation for the discordance between phenotype and genome-wide ancestry, demonstrating how localized introgression can preserve genomic regions associated with phenotypic divergence despite extensive genomic homogenization. This system illustrates how individual genomic regions can retain distinct evolutionary histories long after the surrounding genome has largely homogenized.

evolutionary biology↗

bioRxiv2026

African Campylobacter jejuni genomes reveal globally connected population structure and regionally variable resistance, virulence and mobilome profiles

Campylobacter jejuni is a leading foodborne cause of gastroenteritis, but genomic surveillance remains uneven across Africa. A recent East Africa study combined whole-genome sequencing and antimicrobial susceptibility testing for Campylobacter isolates from humans with diarrhea in Kenya and poultry in Tanzania, showing high sequence-type diversity and substantially higher multidrug resistance in poultry. We extended this regional evidence by analyzing 1,013 publicly available C. jejuni genomes, including 718 African and 295 non-African comparator genomes, with standardized assembly, genotyping, phylogenomics, pangenome reconstruction, antimicrobial resistance, virulence, and mobile-element profiling. African genomes were geographically concentrated but genetically diverse, included globally distributed and regionally enriched lineages, and showed an open pangenome dominated by low-frequency gene families. Resistance and virulence determinants were unevenly distributed by region and lineage. These findings place African C. jejuni diversity within a global evolutionary framework and support expanded, integrated One Health genomic surveillance. Data summaryAll genome sequence data analysed in this study were retrieved from publicly accessible repositories, including the National Center for Biotechnology Information Sequence Read Archive and Assembly resources and corresponding records available through the International Nucleotide Sequence Database Collaboration where applicable. Accession identifiers, BioSample records, run accessions, country metadata and host/source information for all analysed genomes are provided in the combined Supplementary Data workbook. No new sequence data were generated. The analysis used publicly available data generated by other investigators, and the original data-generating studies are cited where appropriate. Derived analytical outputs supporting the findings are included in the manuscript and Supplementary Information. Impact statementGenomic surveillance of Campylobacter jejuni remains uneven globally, and African data are still underrepresented in many comparative analyses. This study brings together publicly available African and non-African C. jejuni genomes in a single standardized comparative framework, linking population structure, pangenome composition, antimicrobial-resistance determinants, virulence-associated loci and mobile-element profiles. The work shows that African C. jejuni diversity is not peripheral to the global population: African genomes include globally distributed sequence types, regionally enriched lineages and a large accessory-gene repertoire. By separating genomic surveillance signals from population-representative prevalence claims, the study provides a cautious framework for interpreting public genome collections from settings with unequal sampling. The findings support broader One Health genomic surveillance, improved metadata completeness and geographically balanced sequencing to better understand foodborne transmission, resistance evolution and lineage diversification in this important zoonotic pathogen.

microbiology↗

bioRxiv2026

The chromosome-scale genome of the pine bark adelgid reveals conserved macrosynteny across Adelgidae

Chromosome evolution varies widely across Aphidomorpha: several aphid lineages exhibit extensive interautosomal reshuffling, whereas comparisons involving grape phylloxera and Adelges suggest greater chromosome conservation outside Aphididae. However, all published adelgid genomes represent the genus Adelges, leaving conservation across deeper adelgid divergences unresolved. Here, we report a chromosome-scale genome for the pine bark adelgid, Pineus strobi, providing the first genome for the genus Pineus and extending genomic sampling to an early-diverging adelgid lineage. Synteny analyses revealed broad conservation of major linkage groups between Pineus and Adelges across approximately 90 million years. The five grape phylloxera chromosomes also showed broad correspondence to the ten adelgid chromosomes, consistent with a limited number of chromosomal fusions or fissions and relatively little exchange among major linkage groups. These findings strengthen evidence that such extensive interautosomal reshuffling is not characteristic of Aphidomorpha as a whole. We also recovered a complete 25.3 kb mitochondrial genome with expanded, repeat-rich noncoding regions and complete circular genomes for the obligate nutritional symbionts "Candidatus Annandia pinicola" and "Candidatus Hartigia pinicola." Comparisons with Pineus similis symbionts revealed conserved coding capacity and genome-wide synteny, supporting conservation of this dual nutritional symbiosis in pine-associated adelgids. Finally, we recovered the first complete Wolbachia genome reported from an adelgid. Because Wolbachia can induce parthenogenesis, its presence in P. strobi raises the possibility that it contributes to the maintenance or reinforcement of parthenogenesis in a species lacking a viable sexual generation. Together, these genomes provide an integrated resource for aphidomorph evolution and symbiosis. Significance statementSeveral aphid lineages exhibit extensive reshuffling among autosomes, whereas previous comparisons with grape phylloxera and Adelges cooleyi suggested greater chromosome conservation outside Aphididae; however, all published adelgid genomes have represented Adelges, leaving the long-term stability of chromosome organization across Adelgidae unresolved. The first chromosome-scale genome from Pineus reveals broad conservation of major linkage groups across approximately 90 million years of adelgid evolution and clear correspondence with the five chromosomes of the grape phylloxera, supporting chromosome conservation outside Aphididae. The accompanying mitochondrial and symbiont genomes provide an integrated resource for studying aphidomorph genome evolution and symbiosis.

evolutionary biology↗

bioRxiv2015

Direct estimate of the spontaneous mutation rate uncovers the effects of drift and recombination in the Chlamydomonas reinhardtii plastid genome

Plastids perform crucial cellular functions, including photosynthesis, across a wide variety of eukaryotes. Since endosymbiosis, plastids have maintained independent genomes that now display a wide diversity of gene content, genome structure, gene regulation mechanisms, and transmission modes. The evolution of plastid genomes depends on an input of de novo mutation, but our knowledge of mutation in the plastid is limited to indirect inference from patterns of DNA divergence between species. Here, we use a mutation accumulation experiment, where selection acting on mutations is rendered ineffective, combined with whole-plastid genome sequencing to directly characterize de novo mutation in Chlamydomonas reinhardtii. We show that the mutation rates of the plastid and nuclear genomes are similar, but that the base spectra of mutations differ significantly. We integrate our measure of the mutation rate with a population genomic dataset of 20 individuals, and show that the plastid genome is subject to substantially stronger genetic drift than the nuclear genome. We also show that high levels of linkage disequilibrium in the plastid genome are not due to restricted recombination, but are instead a consequence of increased genetic drift. One likely explanation for increased drift in the plastid genome is that there are stronger effects of genetic hitchhiking. The presence of recombination in the plastid is consistent with laboratory studies in C. reinhardtii and demonstrates that although the plastid genome is thought to be uniparentally inherited, it recombines in nature at a rate similar to the nuclear genome.

Genomics↗

bioRxiv2016

FGMP: assessing fungal genome completeness and gene content

BackgroundInexpensive high-throughput DNA sequencing has democratized access to genetic information for most organisms so that research utilizing a genome or transcriptome of an organism is not limited to model systems. However, the quality of the assemblies of sampled genomes can vary greatly which hampers utility for comparisons and meaningful interpretation. The uncertainty of the completeness of a given genome sequence can limit feasibility of asserting patterns of high rates of gene loss reported in many lineages.\n\nResultsWe propose a computational framework and sequence resource for assessing completeness of fungal genomes called FGMP (Fungal Genome Mapping Project). Our approach is based on evolutionary conserved sets of proteins and DNA elements and is applicable to various types of genomic data. We present a comparison of FGMP and state-of-the-art methods for genome completeness assessment utilizing 246 genome assemblies of fungi. We discuss genome assembly improvements/degradations in 57 cases where assemblies have been updated, as recorded by NCBI assembly archive.\n\nConclusionFGMP is an accurate tool for quantifying level of completion from fungal genomic data. It is particularly useful for non-model organisms without reference genomes and can be used directly on unassembled reads, which can help reducing genome sequencing costs.

Genomics↗

bioRxiv2020

Regional genomic heritability mapping for agronomic traits in sugarcane

BackgroundThe identification of genomic regions involved in agronomic traits is the primary concern for sugarcane breeders. Genome-wide association studies (GWAS) leverage the sequence variations to bridge phenotypes and genotypes. However, their effectiveness is limited in species with high ploidy and large genomes, such as sugarcane. As an alternative, a regional heritability mapping (RHM) method can be used to capture genetic signals that may be missed by GWAS by combining genetic variance from neighboring regions. We used RHM to screen the sugarcane genome aiming to identify regions with higher heritability associated with agronomic traits. We considered percentage of fiber in sugarcane bagasse (FB), apparent percentage of sugarcane sucrose (PC), tonnes of pol per hectare (TPH), and tonnes of stalks per hectare (TSH). MethodsSequence-capture data of 508 sugarcane (Saccharum spp.) clones from a breeding population under selection were processed for variant calling analysis using the sugarcane genome cultivar R570 as a reference. A set of 375,195 single nucleotide polymorphisms were selected after quality control. RHM was conducted by splitting the sugarcane genome into windows of 2 Mb length. ResultsWe selected the windows explaining > 20% of the total genomic heritability for TPH (64 windows - 5,654 genes) and TSH (72 windows - 6,050 genes), and > 15% for PC (16 windows - 1,517 genes) and FB (17 windows - 1,615 genes). The top five windows that explained the highest genomic heritability ranged from 20.8 to 24.6% for FB (629 genes), 18.0 to 22.0% for PC (452 genes), 53.8 to 66.0% for TPH (705 genes), and 59.5 to 67.4% for TSH (413 genes). The functional annotation of genes included in those top five windows revealed a set of genes that encode enzymes that integrate carbon metabolism, starch and sucrose metabolism, and phenylpropanoid biosynthesis pathways. ConclusionsThe selection of windows that explained the large proportions of genomic heritability allowed us to identify genomic regions containing a set of genes that are related to the agronomic traits in sugarcane. These windows spanned a region of 58.38Mb, which corresponds to 14.28% of the reference assembly in the sugarcane genome. We contend that RHM can be used as an alternative method for sugarcane breeders to reduce the complexity of the sugarcane genome.

genomics↗

bioRxiv2020

Dynamics of transposable elements in recently diverged fungal pathogens: lineage-specific transposable element content and efficiency of genome defences

Transposable elements (TEs) impact genome plasticity, architecture and evolution in fungal plant pathogens. The wide range of TE content observed in fungal genomes reflects diverse efficacy of host-genome defence mechanisms that can counter-balance TE expansion and spread. Closely related species can harbour drastically different TE repertoires, suggesting variation in the efficacy of genome defences. The evolution of fungal effectors, which are crucial determinants of pathogenicity, has been linked to the activity of TEs in pathogen genomes. Here we describe how TEs have shaped genome evolution of the fungal wheat pathogen Zymoseptoria tritici and four closely related species. We compared de novo TE annotations and Repeat-Induced Point mutation signatures in thirteen genomes from the Zymoseptoria species-complex. Then, we assessed the relative insertion ages of TEs using a comparative genomics approach. Finally, we explored the impact of TE insertions on genome architecture and plasticity. The thirteen genomes of Zymoseptoria species reflect different TE dynamics with a majority of recent insertions. TEs associate with distinct genome compartments in all Zymoseptoria species, including chromosomal rearrangements, genes showing presence/absence variation and effectors. European Z. tritici isolates have reduced signatures of Repeat-Induced Point mutations compared to Iranian isolates and closely related species. Our study supports the hypothesis that ongoing but moderate TE mobility in Zymoseptoria species shapes pathogen genome evolution.

genomics↗

bioRxiv2021

Benchmarking Oxford Nanopore read assemblers for high-quality molluscan genomes

Choosing the optimum assembly approach is essential to achieving a high-quality genome assembly suitable for comparative and evolutionary genomic investigations. Significant recent progress in long-read sequencing technologies such as PacBio and Oxford Nanopore Technologies (ONT) also brought about a large variety of assemblers. Although these have been extensively tested on model species such as Homo sapiens and Drosophila melanogaster, such benchmarking has not been done in Mollusca which lacks widely adopted model species. Molluscan genomes are notoriously rich in repeats and are often highly heterozygous, making their assembly challenging. Here, we benchmarked 10 assemblers based on ONT raw reads from two published molluscan genomes of differing properties, the gastropod Chrysomallon squamiferum (356.6Mb, 1.59% heterozygosity) and the bivalve Mytilus coruscus (1593Mb, 1.94% heterozygosity). By optimising the assembly pipeline, we greatly improved both genomes from previously published versions. Our results suggested that 40-50X of ONT reads are sufficient for high-quality genomes, with Flye being the recommended assembler for compact and less heterozygous genomes exemplified by C. squamiferum, while NextDenovo excelled for more repetitive and heterozygous molluscan genomes exemplified by M. coruscus. A phylogenomic analysis utilising the two updated genomes with other 32 published high-quality lophotrochozoan genomes resulted in maximum support across all nodes, and we show that improved genome quality also leads to more complete matrices for phylogenomic inferences. Our benchmarking will ensure the efficiency in future assemblies for molluscs and perhaps also other marine phyla with few genomes available.

genomics↗

bioRxiv2021

Investigating the impact of reference assembly choice on genomic analyses in a cattle breed

BackgroundReference-guided read alignment and variant genotyping are prone to reference allele bias, particularly for samples that are greatly divergent from the reference genome. A Hereford-based assembly is the widely accepted bovine reference genome. Haplotype-resolved genomes that exceed the current bovine reference genome in quality and continuity have been assembled for different breeds of cattle. Using whole genome sequencing data of 161 Brown Swiss cattle, we compared the accuracy of read mapping and sequence variant genotyping as well as downstream genomic analyses between the bovine reference genome (ARS-UCD1.2) and a highly continuous Angus-based assembly (UOA_Angus_1). ResultsRead mapping accuracy did not differ notably between the ARS-UCD1.2 and UOA_Angus_1 assemblies. We discovered 22,744,517 and 22,559,675 high-quality variants from ARS-UCD1.2 and UOA_Angus_1, respectively. The concordance between sequence- and array-called genotypes was high and the number of variants deviating from Hardy-Weinberg proportions was low at segregating sites for both assemblies. More artefactual INDELs were genotyped from UOA_Angus_1 than ARS-UCD1.2 alignments. Using the composite likelihood ratio test, we detected 40 and 33 signatures of selection from ARS-UCD1.2 and UOA_Angus_1, respectively, but the overlap between both assemblies was low. Using the 161 sequenced Brown Swiss cattle as a reference panel, we imputed sequence variant genotypes into a mapping cohort of 30,499 cattle that had microarray-derived genotypes. The accuracy of imputation (Beagle R2) was very high (0.87) for both assemblies. Genome-wide association studies between imputed sequence variant genotypes and six dairy traits as well as stature produced almost identical results from both assemblies. ConclusionsThe ARS-UCD1.2 and UOA_Angus_1 assemblies are suitable for reference-guided genome analyses in Brown Swiss cattle. Although differences in read mapping and genotyping accuracy between both assemblies are negligible, the choice of the reference genome has a large impact on detecting signatures of selection using the composite likelihood ratio test. We developed a workflow that can be adapted and reused to compare the impact of reference genomes on genome analyses in various breeds, populations and species.

genomics↗

bioRxiv2021

A chromosome-level genome assembly of the European Beech (Fagus sylvativa) reveals anomalies for organelle DNA integration, repeat content and distribution of SNPs

The European Beech is the dominant climax tree in most regions of Central Europe and valued for its ecological versatility and hardwood timber. Even though a draft genome has been published recently, higher resolution is required for studying aspects of genome architecture and recombination. Here we present a chromosome-level assembly of the more than 300 year-old reference individual, Bhaga, from the Kellerwald-Edersee National Park (Germany). Its nuclear genome of 541 Mb was resolved into 12 chromosomes varying in length between 28 Mb and 73 Mb. Multiple nuclear insertions of parts of the chloroplast genome were observed, with one region on chromosome 11 spanning more than 2 Mb of the genome in which fragments up to 54,784 bp long and covering the whole chloroplast genome were inserted randomly. Unlike in Arabidopsis thaliana, ribosomal cistrons are present in Fagus sylvatica only in four major regions, in line with FISH studies. On most assembled chromosomes, telomeric repeats were found at both ends, while centromeric repeats were found to be scattered throughout the genome apart from their main occurrence per chromosome. The genome- wide distribution of SNPs was evaluated using a second individual from Jamy Nature Reserve (Poland). SNPs, repeat elements and duplicated genes were unevenly distributed in the genomes, with one major anomaly on chromosome 4. The genome presented here adds to the available highly resolved plant genomes and we hope it will serve as a valuable basis for future research on genome architecture and for understanding the past and future of European Beech populations in a changing climate.

genomics↗

bioRxiv2021

A chromosome-level genome assembly and annotation of a maize elite breeding line Dan340

BackgroundMaize is not only one of the most important crops grown worldwide for food, forage, and biofuel, but also an important model organism for fundamental research in genetics and genomics. Owing to its importance in crop science, genetics and genomics, several reference genomes of common maize inbred line (genetic material) have been released, but some genomes of important genetic germplasm resources in maize breeding research are still lacking. The maize cultivar Dan340 is an excellent backbone inbred line of the Luda Red Cob Group with several desirable characteristics, such as disease resistance, lodging resistance, high combining ability, and wide adaptability. FindingsIn this study, we constructed a high-quality chromosome-level reference genome for Dan340 by combining PacBio long HiFi sequencing reads, Illumina short reads and chromosomal conformational capture (Hi-C) sequencing reads. The final assembly of the Dan340 genome was 2,348.72 Mb, including 2,738 contigs and 2,315 scaffolds with N50 of 41.49 Mb and 215.35 Mb, respectively. Repeat sequences accounted for 73.40% of the genome size and 39,733 protein-coding genes were annotated. Analysis of genes in the Dan340 genome, together with those from B73, Mo17 and SK, were clustered into 27,654 gene families. There were 1,806 genes from 359 gene families that were specific to Dan340, of which many had functional gene ontology annotations relating to "porphyrin-containing compound metabolic process", "tetrapyrrole biosynthetic process", and "tetrapyrrole metabolic process". ConclusionsThe completeness and continuity of the genome were comparable to those of other important maize inbred lines. The assembly and annotation of this genome not only facilitates our understanding about of intraspecific genome diversity in maize, but also provides a novel resource for maize breeding improvement. Research AreasGenetics and Genomics; Agriculture, Plant Genetics Data Description

genomics↗

bioRxiv2021

Rapid Genomic Characterization and Global Surveillance of Klebsiella Using Pathogenwatch

BackgroundKlebsiella species, including the notable pathogen K. pneumoniae, are increasingly associated with antimicrobial resistance (AMR). Genome-based surveillance can inform interventions aimed at controlling AMR. However, its widespread implementation requires tools to streamline bioinformatic analyses and public health reporting. MethodsWe developed the web application Pathogenwatch, which implements analytics tailored to Klebsiella species for integration and visualization of genomic and epidemiological data. We populated Pathogenwatch with 16,537 public Klebsiella genomes to enable contextualization of user genomes. We demonstrated its features with 1,636 genomes from four low- and middle-income countries (LMICs) participating in the NIHR Global Health Research Unit (GHRU) on AMR. ResultsUsing Pathogenwatch, we found that GHRU genomes were dominated by a small number of epidemic drug-resistant clones of K. pneumoniae. However, differences in their distribution were observed (e.g. ST258/512 dominated in Colombia, ST231 in India, ST307 in Nigeria, ST147 in the Philippines). Phylogenetic analyses including public genomes for contextualization enabled retrospective monitoring of their spread. In particular, we identified hospital outbreaks, detected introductions from abroad, and uncovered clonal expansions associated with resistance and virulence genes. Assessment of loci encoding O-antigens and capsule in K. pneumoniae, which represent possible vaccine candidates, showed that three O-types (O1-O3) represented 88.9% of all genomes, whereas capsule types were much more diverse. ConclusionsPathogenwatch provides a free, accessible platform for real-time analysis of Klebsiella genomes to aid surveillance at local, national and global levels. We have improved representation of genomes from GHRU participant countries, further facilitating ongoing surveillance. summaryPathogenwatch is a free web-application for analysis of Klebsiella genomes to aid surveillance at local, national and global levels. We improved the representation of genomes from middle-income countries through the Global Health Research Unit on AMR, further facilitating ongoing surveillance.

genomics↗

bioRxiv2021

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

BackgroundAn 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 beetles 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. ResultsHigh-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 = 99.11%). These quality metrics place our assembly as the most complete of the published Coleopteran genomes. The structural annotation of the nuclear genome assembly contained a highly-accurate set of 16,371 protein-coding genes showing BUSCO completeness of 92.09%, as well as the expected number of non-coding RNAs and the number and structure of paralogous genes in a gene family like Sigma GST. ConclusionsThe 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.

genomics↗

bioRxiv2021

Comparative Analysis and Data Provenance for 1,113 Bacterial Genome Assemblies

The quality and traceability of microbial genomics data in public databases is deteriorating as they rapidly expand and struggle to cope with data curation challenges. While the availability of public genomic data has become essential for modern life sciences research, the curation of the data is a growing area of concern that has significant real-world impacts on public health epidemiology, drug discovery, and environmental biosurveillance research1-6. While public microbial genome databases such as NCBIs RefSeq database leverage the scalability of crowd sourcing for growth, they do not require data provenance to the original biological source materials or accurate descriptions of how the data was produced7. Here, we describe the de novo assembly of 1,113 bacterial genome references produced from authenticated materials sourced from the American Type Culture Collection (ATCC), each with full data provenance. Over 98% of these ATCC Standard Reference Genomes (ASRGs) are superior to assemblies for comparable strains found in NCBIs RefSeq database. Comparative genomics analysis revealed significant issues in RefSeq bacterial genome assemblies related to genome completeness, mutations, structural differences, metadata errors, and gaps in traceability to the original biological source materials. For example, nearly half of RefSeq assemblies lack details on sample source information, sequencing technology, or bioinformatics methods. We suggest there is an intrinsic connection between the quality of genomic metadata, the traceability of the data, and the methods used to produce them with the quality of the resulting genome assemblies themselves. Our results highlight common problems with " reference genomes" and underscore the importance of data provenance for precision science and reproducibility. These gaps in metadata accuracy and data provenance represent an " elephant in the room" for microbial genomics research, but addressing these issues would require raising the level of accountability for data depositors and our own expectations of data quality.

genomics↗

bioRxiv2022

The nature and chromosomal landscape of endogenous retroviruses (ERVs) integrated in the sheep nuclear genome

Endogenous retroviruses (ERVs) represent genomic components of retroviral origin that are found integrated in the genomes of various species of vertebrates. These genomic elements have been widely characterized in model organisms and humans. However, composition and abundances of ERVs have not been categorized fully in all domestic animals. The advent of next generation sequencing technologies, development of bioinformatics tools, availability of genomic databases and molecular cytogenetic techniques have revolutionized the exploration of the genome structure. Here, we investigated the nature, abundance, organization and assembly of ERVs and complete genomes of Jaagsiekte sheep retrovirus (JSRV) from high-throughput sequencing (HTS) data from two Iraqi domestic sheep breeds. We used graph-based read clustering (RepeatExplorer), frequency analysis of short motifs (k-mers), alignment to reference genome assemblies and fluorescent in situ hybridization (FISH). Three classes of ERVs were identified with the total genomic proportions of 0.55% from all analyzed whole genome sequencing raw reads, while FISH to ovine metaphase chromosomes exhibited abundant centromeric to dispersed distribution of these ERVs. Furthermore, the complete genomes of JSRV of two Iraqi sheep breeds were assembled and phylogenetically clustered with the known enJSRV proviruses in sheep worldwide. Characterization of partial and complete sequences of mammalian ERVs is valuable to provide insights into the genome landscape, to help with future genome assemblies and to identify potential sources of disease when ERVs become active.

genomics↗

bioRxiv2022

A beary good genome: Haplotype-resolved, chromosome-level assembly of the brown bear (Ursus arctos)

The brown bear (Ursus arctos) is the second largest and most widespread extant terrestrial carnivore on Earth and has recently emerged as a medical model for human metabolic diseases. Here, we report a fully-phased chromosome-level assembly of a male North American brown bear built by combining Pacific Biosciences (PacBio) HiFi data and publicly available Hi-C data. The final genome size is 2.47 Gigabases (Gb) with a scaffold and contig N50 length of 70.08 and 43.94 Mb, respectively. BUSCO analysis revealed that 94.5% of single-copy orthologs from mammalia were present in the genome (the highest of any ursid genome to date). Repetitive elements accounted for 44.48% of the genome and a total of 20,480 protein coding genes were identified. Based on whole genome alignment, the brown bear is highly syntenic with the polar bear, and our phylogenetic analysis of 7,246 single-copy BUSCOs supports the currently proposed species tree for Ursidae. This highly contiguous genome assembly will support future research on both the evolutionary history of the bear family and the physiological mechanisms behind hibernation, the latter of which has broad medical implications. SignificanceBrown bears (Ursus arctos) are the most widespread, large terrestrial carnivore on the planet and represent an interesting example of speciation through hybridization, as well as a medical model for sedentary lifestyle-related disease. Although a previous genome for a brown bear has been published, the reported contig N50 was low (only [~]530 kb), despite being scaffolded into putative chromosomes. Genomes of this quality limit the accuracy of analyses which rely on long contiguous stretches of the genome to be assembled (such as with many demographic analyses) as well as attempts at connecting genotype to phenotype (such as in association analyses). In order to support studies on both the complex hybridization history of the brown bear and investigations into medically-relevant phenotypes, we generated a fully-phased, chromosome-level assembly from a male grizzly bear. The genome has a total size of 2.47 Gb and 90% of the genome is contained in 36 scaffolds, roughly corresponding to one autosome per scaffold. This high-quality genome will enable studies across a variety of disciplines, including conservation, evolution, and medicine.

genomics↗

bioRxiv2022

Effect of reduced genomic representation on using runs of homozygosity for inbreeding characterization

Runs of homozygosity (ROHs) are proxy for genomic Identical-by-Descent segments and are increasingly used to measure individual inbreeding. ROHs analyses are mostly carried out on SNPs-arrays and whole-genome-sequencing data. Softwares recurrently used for their detection usually assume that genomic positions which have not been genotyped are non-variant. This might be true for whole-genome-sequencing data, but not for reduced genomic representations and can lead to spurious ROHs detection. We simulated the outputs of whole-genome-sequencing, two SNP-arrays and RAD-sequencing for three populations with different sizes. We compare the results of ROHs calling with two softwares: PLINK and RZooRoH. We demonstrate that to obtain meaningful estimates of inbreeding coefficients, RZooRoH requires fraction of genome seven times smaller compared to PLINK. When the SNP density is above 20 SNPs/Mb for PLINK and 3 SNPs/Mb for RZooRoH, ranks of ROHs-based inbreeding coefficients are conserved among individuals. With reduced genomic representations, ROHs distributions are consistently biased towards an underestimation of the total numbers of small and an overestimation of the total numbers of large ROHs, except for RZooRoH and high-density SNPs-arrays. We conclude that both ROHs-based inbreeding coefficients and ROHs distributions exact quantification are highly dependent on the fraction of genome sequenced and should thus be treated with caution. However, relative inbreeding estimates, such as comparison between individuals or populations, are reliable with reduced genomic representations providing that the fraction of genome sequenced is large enough. Consequently, we advise researchers working with reduced genomic data to use SNPs-independent measures or model-based ROHs calling methods for inbreeding estimations.

genomics↗