Genome Assembly Research Articles

Orthopoxvirus Genome Sequencing, Assembly, and Analysis.

Poxviruses have exceptionally large genomes compared to most other viruses, which represent unique challenges to sequencing and assembly due to complex features such as repeat elements and low complexity sequences. The 2022 global mpox outbreak led to an unprecedented level of poxvirus sequencing as public health and research institutions faced with large sample numbers and demand for fast turnaround, merged NGS protocols designed for small RNA viruses with poxvirus expertise. Traditional manual assembly, checking, and editing of genomes was not feasible. Here, we present a protocol for metagenomic sequencing and orthopoxvirus genome assembly directly from DNA extracted from a patient lesion swab with no viral enrichment or host depletion. This sequencing approach is cost effective when using high throughput sequencing instruments and allows for detection of genomic insertions, deletions, and large rearrangement with confidence. We describe usage of two publicly available bioinformatic pipelines for genome assembly, quality control, annotation, and submission to sequence repositories.

The genome sequence of the pear, Pyrus communis L.

We present a genome assembly from a specimen of Pyrus communis (the pear; Streptophyta; Magnoliopsida; Rosales; Rosaceae). The genome sequence has a total length of 487.30 megabases. Most of the assembly is scaffolded into 17 chromosomal pseudomolecules. The mitochondrial and plastid genome assemblies have lengths of 443.53 kilobases and 159.93 kilobases, respectively. Gene annotation of this assembly on Ensembl identified 37,713 protein-coding genes.

The genome sequence of the straw spear-moss, Straminergon stramineum (Dicks. ex Brid.) Hedenas, 1993

We present a genome assembly from a haploid Straminergon stramineum gametophyte (the straw spear-moss; Streptophyta; Bryopsida; Hypnales; Calliergonaceae). The genome sequence spans 326.30 megabases. Most of the assembly is scaffolded into 11 chromosomal pseudomolecules. The mitochondrial and plastid genome assemblies have lengths of 104.6 kilobases and 124.69 kilobases, respectively.

The genome sequence of the crisped pincushion, Ulota crispa (Hedw.) Brid.

We present a genome assembly from a haploid Ulota crispa gametophyte (the crisped pincushion; Streptophyta; Bryopsida; Orthotrichales; Orthotrichaceae). The genome sequence spans 275.00 megabases. Most of the assembly is scaffolded into 11 chromosomal pseudomolecules. The mitochondrial and plastid genome assemblies have lengths of 104.64 kilobases and 123.54 kilobases, respectively.

The genome sequence of wych elm, Ulmus glabra Huds.

We present a genome assembly from a diploid specimen of Ulmus glabra (wych elm; Streptophyta; Magnoliopsida; Rosales; Ulmaceae). The assembly contains two haplotypes with total lengths of 1,788.20 megabases and 1,860.20 megabases, respectively. Most of haplotype 1 (97.9%) is scaffolded into 14 chromosomal pseudomolecules. Haplotype 2 was submitted as a scaffold-level assembly. The mitochondrial and plastid genome assemblies have lengths of 520.46 kilobases and 159.28 kilobases, respectively.

Genomic Insights into Fusarium verticillioides Diversity: The Genome of Two Clinical Isolates and Their Demethylase Inhibitor Fungicides Susceptibility

Fusarium verticillioides is an important plant pathogen in maize and other cereals that is seldom detected as the cause of human fusariosis. Here, we provide the analysis of the available diversity of F. verticillioides sequenced worldwide and report the first two genome assemblies and annotations (including mitochondrial DNA) of Fusarium verticillioides from clinical settings. Fusarium verticillioides 05-0160 (IUM05-0160) and Fusarium verticillioides 09-1037 (IUM09-1037) strains were obtained from the bone marrow and blood of two immunocompromised patients, respectively. The phylogenomic analysis confirmed the species identity of our two strains. Comparative genomic analyses among the reannotated F. verticillioides genomes (n = 46) did not lead to the identification of unique genes specific to the clinical samples. Two subgroups in the F. verticillioides clade were also identified and confirmed by a mitochondrial diversity study. Clinical strains (n = 4) were positioned in the multigene phylogenetic tree without any correlation between the host and the tree branches, grouping with plant-derived strains. To investigate the existence of a potential fitness advantage of our two clinical strains, we compared demethylase inhibitor fungicides susceptibility against the reference Fusarium verticillioides 7600, showing, on average, lower susceptibility to agricultural and medical-used antifungals. A significant reduction in susceptibility was observed for itraconazole and tetraconazole, which might be explained by structural changes in CYP51A and CYP51C sequences. By providing the first two annotated genomes of F. verticillioides from clinical settings comprehensive of their mitogenomes, this study can serve as a base for exploring the fitness and adaptation capacities of Fusarium verticillioides infecting different kingdoms.

Comparative and Adaptive Analyses of the Complete Chloroplast Genome Diversity in Sium serra

Background/Objectives: Sium serra is distributed in Korea, China, and Japan. It was first identified as the genus Pimpinella and then reclassified as Sium by Kitagawa. Some Sium species are used as herbal medicine and are often confused with the similar form Ligusticum sinense. In this study, we analyzed the cp genome of S. serra and conducted comparative analyses with the cp genomes of related taxa. Methods: We extracted gDNA from fresh leaves and sequenced it using Illumina HiSeq2500. For the chloroplast genome assembly, de novo assembly was performed using Velvet v1.2.07. For the annotation, GeSeq and NCBI BLASTN were used. Afterwards, related taxa were analyzed using programs such as DnaSP and MISA. Results: S. serra was excluded from the study on the chloroplast (cp) genome in Sium because it was classified as Pimpinella in China. Therefore, this study aimed to analyze the cp genome of S. serra for the first time and its location within the genus Sium. The complete cp genome of S. serra was 154,755 bp in length, including a pair of inverted repeats, each 26,255 bp, a large single-copy region of 84,581 bp, and a small single-copy region of 17,664 bp. The cp genome comprised 79 protein-coding, 30 tRNA, and 4 rRNA genes. Furthermore, six regions of high nucleotide diversity were identified in the genus Sium. In the genus Sium, 1630 repeats that can serve as markers were also identified. Eight protein-coding genes with high KA/KS values were under positive selection in the Sium. Our phylogenetic analyses suggest that S. serra was positioned with high bootstrap support within the Sium of the tribe Oenantheae, specifically in the southern Palearctic subclade. Conclusions: In this study, the S. serra chloroplast genome was sequenced and assembled. The genus Sium formed a monophyletic group; however, as not all the Sium species were included in this study, further research is necessary. This study can serve as foundational data not only for Sium but also for the tribe Oenantheae.

The genome sequence of a tachinid fly, Gymnocheta viridis (Fallén, 1810)

We present a genome assembly from an individual male specimen of Gymnocheta viridis (tachinid fly; Arthropoda; Insecta; Diptera; Tachinidae). The genome sequence has a total length of 600.30 megabases. Most of the assembly (98.1%) is scaffolded into 6 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 19.34 kilobases in length. Gene annotation of this assembly on Ensembl identified 12,716 protein-coding genes.

MetaCONNET: A metagenomic polishing tool for long-read assemblies.

Accurate and high coverage genome assemblies are the basis for downstream analysis of metagenomic studies. Long-read sequencing technology is an ideal tool to facilitate the assemblies of metagenome, except for the drawback of usually producing reads with high sequencing error rate. Many polishing tools were developed to correct the sequencing error, but most are designed on the ground of one or two species. Considering the complexity and uneven depth of metagenomic study, we present a novel deep-learning polishing tool named MetaCONNET for polishing metagenomic assemblies. We evaluate MetaCONNET against Medaka, CONNET and NextPolish in accuracy, coverage, contiguity and resource consumption. Our results demonstrate that MetaCONNET provides a valuable polishing tool and can be applied to many metagenomic studies.

The genome sequence of Radford’s Flame Shoulder, Ochropleura leucogaster (Freyer, 1831)

We present a genome assembly from an individual male Ochropleura leucogaster (Freyer, 1831) (Radford's Flame Shoulder; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence has a total length of 545.70 megabases. Most of the assembly (99.93%) is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.37 kilobases in length. Gene annotation of this assembly on Ensembl identified 12,155 protein-coding genes.

The genome sequence of a sawfly, Athalia circularis (Klug, 1815)

We present a genome assembly from a female specimen of Athalia circularis (sawfly; Arthropoda; Insecta; Hymenoptera; Athaliidae). The genome sequence has a total length of 201.90 megabases. Most of the assembly (99.53%) is scaffolded into 6 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 17.77 kilobases in length.

The genome sequence of a tachinid fly, Phania funesta (Meigen, 1824)

We present a genome assembly from an individual male tachinid fly, Phania funesta (Arthropoda; Insecta; Diptera; Tachinidae). The genome sequence has a total length of 557.40 megabases. Most of the assembly is scaffolded into 6 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 17.18 kilobases in length.

A high-quality chromosome-level genome assembly of the traditional Chinese medicinal herb Zanthoxylum nitidum

Dried roots of Zanthoxylum nitidum (2n=2x=70, family Rutaceae) was referred as “Liang-Mian-Zhen” in Chinese, acting as a valuable species due to its notable pharmacological activities. Herein, we combined PacBio HiFi data together with Hi-C mapping technology to construct a chromosome-scale reference genome assembly for Z. nitidum. The assembly reached a length of 2.24 Gb, successfully anchoring 99.31% of sequences onto 35 pseudo-chromosomes. Among these, 26 chromosomes achieved telomere-to-telomere assembly, and 11 chromosomes were gap-free. The contigs N50 and scaffolds N50 reached 57.61 Mb and 78.00 Mb, respectively. Transposable elements comprised 81.44% of the Z. nitidum genome, and over 78% of them were long-terminal repeat retrotransposon elements. Furthermore, 32,737 protein-coding genes were identified and 99.38% of all were functionally annotated. The completeness of the genome assembly and final gene sets reached 97.83% and 96.47% based on Benchmarking Universal Single-Copy Orthologs (BUSCO), respectively. Taken together, our results provided a high-quality chromosome-level assembly of Z. nitidum genome and will be a valuable resource that will facilitate breeding varieties with higher alkaloids content.

The rate and spectrum of new mutations in mice inferred by long-read sequencing

All forms of genetic variation originate from new mutations, making it crucial to understand their rates and mechanisms. Here, we use long-read PacBio sequencing to investigate de novo mutations that accumulated in 12 inbred mouse lines derived from three commonly used inbred strains (C3H, C57BL/6, and FVB) maintained for 8-15 generations in a mutation accumulation (MA) experiment. We built chromosome-level genome assemblies based on the MA line founders' genomes, and then employed a combination of read and assembly-based methods to call the complete spectrum of new mutations. On average, there are ~45 mutations per haploid genome per generation, about half of which (54%) are insertions and deletions shorter than 50 bp (indels). The remainder are single nucleotide mutations (SNMs, 44%) and large structural mutations (SMs, 2%). We found that the degree of DNA repetitiveness is positively correlated with SNM and indel rates, and that a substantial fraction of SMs can be explained by homology-dependent mechanisms associated with repeat sequences. Most (90%) indels can be attributed to microsatellite contractions and expansions, and there is a marked bias towards 4 bp indels. Among the different types of SMs, tandem repeat mutations have the highest mutation rate, followed by insertions of transposable elements (TEs). We uncover a rich landscape of active TEs, and notable differences in their spectrum among MA lines and strains, and a high rate of gene retroposition. Our study offers novel insights into mammalian genome evolution, and highlights the importance of repetitive elements in shaping genomic diversity.

The genome sequence of Common Haircap, Polytrichum commune Hedw. (Polytrichaceae)

We present a genome assembly from a specimen of Polytrichum commune (Common Haircap; Streptophyta; Polytrichopsida; Polytrichales; Polytrichaceae). The genome sequence has a total length of 407.90 megabases. Most of the assembly is scaffolded into 7 chromosomal pseudomolecules. The organelle genomes have also been assembled: the mitochondrial genome is 114.83 kilobases and the plastid genome is 126.25 kilobases in length

The genome sequence of the Welsh wave moth, Venusia cambrica Curtis, 1839

We present a genome assembly from an individual male Venusia cambrica (the Welsh Wave; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence spans 470.40 megabases. Most of the assembly is scaffolded into 38 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 16.44 kilobases in length. Gene annotation of this assembly on Ensembl identified 17,931 protein-coding genes.

The genome sequence of the Brown Heath Robberfly, Tolmerus cingulatus (Fabricius, 1781)

We present a genome assembly from an individual male Tolmerus cingulatus (Brown Heath Robberfly; Arthropoda; Insecta; Diptera; Asilidae). The genome sequence has a total length of 280.00 megabases. Most of the assembly (88.86%) is scaffolded into 6 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 20.2 kilobases in length. Gene annotation of this assembly on Ensembl identified 12,047 protein-coding genes.

Beyond Asexual: Genomics-Driven Progress in Unveiling Sexual Reproduction in Cereal Rust Fungi.

Recent advances in genomics technologies have revolutionized our understanding of cereal rust fungi, providing unprecedented insights into the complexities of their sexual life cycle. Genomic approaches, including long-read sequencing, genome assembly, and haplotype phasing technologies, have revealed critical insights into mating systems, genetic diversity, virulence evolution, and host adaptation. Population genomics studies have uncovered diverse reproductive strategies across different cereal rust species and geographic regions, highlighting the interplay between sexual recombination and asexual reproduction. Transcriptomics have begun to unravel the gene expression networks driving sexual reproduction, while complementary omics approaches such as proteomics, and metabolomics offer potential insights into the underlying molecular processes. Despite this progress, many aspects of rust sexual reproduction remain elusive. Integrating multiple omics approaches with advanced cell biology techniques can help address these knowledge gaps, particularly in understanding sexual reproduction and its role in pathogen evolution. This comprehensive approach will be crucial for developing more targeted and resilient crop protection strategies, ultimately contributing to global food security.

Convergent evolution of berberine biosynthesis.

Berberine is an effective antimicrobial and antidiabetic alkaloid, primarily extracted from divergent botanical lineages, specifically Coptis (Ranunculales, early-diverging eudicot) and Phellodendron (Sapindales, core eudicot). In comparison with its known pathway in Coptis species, its biosynthesis in Phellodendron species remains elusive. Using chromosome-level genome assembly, coexpression matrix, and biochemical assays, we identified six key steps in berberine biosynthesis from Phellodendron amurense, including methylation, hydroxylation, and berberine bridge formation. Notably, we discovered a specific class of O-methyltransferases (NOMT) responsible for N-methylation. Structural analysis and mutagenesis of PaNOMT9 revealed its unique substrate-binding conformation. In addition, unlike the classical FAD-dependent berberine bridge formation in Ranunculales, Phellodendron uses a NAD(P)H-dependent monooxygenase (PaCYP71BG29) for berberine bridge formation, originating from the neofunctionalization of tryptamine 5-hydroxylase. Together, these findings reveal the convergence of berberine biosynthesis between Coptis and Phellodendron and signify the role of the convergent evolution in plant specialized metabolisms.

Masu salmon species complex relationships and sex chromosomes revealed from analyses of the masu salmon (Oncorhynchus masou masou) genome assembly.

Masu salmon (Oncorhynchus masou) are the only Pacific salmon endemic to Asia. Some researchers prefer to categorize these salmon into four subspecies (masu - O. m. masou, amago - O. m. ishikawae, Biwa - O. m. subsp., and Formosan - O. m. formosanus), while others prefer individual species designations. Even though the masu salmon fishery is thousands of years old, classification of the diversity within the masu salmon species complex remains elusive. In this study, a genetic map and reference genome assembly were generated for one species/subspecies (masu) to provide resources for understanding the species complex. In O. m. masou, the sex chromosome was determined to be chromosome 7. Resequenced genomes from two other putative subspecies (amago and Biwa) provided evidence that they do not share the same sex chromosome. Principal component and admixture analyses clustered the amago and Biwa salmon close together. This supported previous findings of a close relationship between amago and Biwa salmon and a more distant relationship to masu salmon for both. Additional analyses of the masu salmon species complex will benefit from using the new reference genome assembly.