Current Genome Research Articles

Whole-genome sequencing and genomic analysis of four Akkermansia strains newly isolated from human feces

BackgroundNumerous studies have demonstrated that Akkermansia is closely associated with human health. These bacteria colonize the mucus layer of the gastrointestinal tract and utilize mucin as their sole source of carbon and nitrogen. Akkermansia spp. exhibit potential as probiotics under specific conditions. However, the gene accumulation curve derived from pan-genome analysis suggests that the genome of Akkermansia strains remains open. Consequently, current genome mining efforts are insufficient to fully capture the intraspecific and interspecific characteristics of Akkermansia, necessitating continuous exploration of the genomic and phenotypic diversity of new isolates.MethodsBased on this finding, we sequenced, assembled, and functionally annotated the whole genomes of four new human isolates from our laboratory: AKK-HX001, AKK-HX002, AKK-HX003, and AKK-HX004.ResultsPhylogenetic analysis revealed that all four isolates belonged to the AmII phylogroup, whereas the type strain DSM 22959 is classified within the AmI phylogroup. Moreover, 2,184 shared homologous genes were identified among the four isolates. Functional annotation using the COG, KEGG, and CAZy databases indicated that the functional genes of the four isolates were primarily associated with metabolism. Two antibiotic resistance genes were identified in AKK-HX001 and AKK-HX002, while three resistance genes were detected in AKK-HX003 and AKK-HX004. Additionally, each of the four isolates possessed two virulence genes and three pathogenicity genes, none of which were associated with pathogenicity. The prediction of mobile genetic elements indicated unequal distributions of GIs among the isolates, and a complete CRISPR system was identified in all isolates except AKK-HX003. Two annotated regions of secondary metabolite biosynthesis genes, both belonging to Terpene, were detected using the antiSMASH online tool.ConclusionThese findings indicate that the four Akkermansia isolates, which belong to a phylogroup distinct from the model strain DSM 22959, exhibit lower genetic risk and may serve as potential probiotic resources for future research.

The complete telomere-to-telomere sequence of a mouse genome.

The current reference genome of Mus musculus, GRCm39, has major gaps in both euchromatic and heterochromatic regions associated with repetitive sequences. In this work, we have sequenced and assembled the telomere-to-telomere genome of mouse haploid embryonic stem cells. The results reveal more than 7.7% of previously uncovered sequences of the mouse genome, including ribosomal DNA arrays and pericentromeric and subtelomeric regions, as well as an additional 140 genes predicted to be protein-coding. This study helps to address knowledge gaps in the mouse genome.

Efficient small fragment sequencing of human, cattle, and bison miRNA, small RNA, or csRNA-seq libraries using AVITI

BackgroundNext-Generation Sequencing (NGS) catalyzed breakthroughs across various scientific domains. Illumina’s sequencing by synthesis method has long been central to NGS, but new sequencing methods like Element Biosciences’ AVITI technology are emerging. AVITI is reported to offer improved signal-to-noise ratios and cost reductions. However, its reliance on rolling circle amplification, which can be affected by polymer size, raises questions about its effectiveness in sequencing small RNAs (sRNAs) such as microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), and many others. These sRNAs are crucial regulators of gene expression and involved in various biological processes. Additionally, capturing capped small RNAs (csRNA-seq) is a powerful method for mapping active or “nascent” RNA polymerase II transcription initiation in tissues and clinical samples.ResultsHere, we report a new protocol for seamlessly sequencing short fragments on the AVITI and demonstrate that AVITI and Illumina sequencing technologies equivalently capture human, cattle (Bos taurus), and bison (Bison bison) sRNA or csRNA sequencing libraries, increasing confidence in both sequencing approaches. Additionally, analysis of generated nascent transcription start site (TSS) data for cattle and bison revealed inaccuracies in their current genome annotations, underscoring the potential and necessity to translate small and nascent RNA sequencing methodologies to livestock.ConclusionsOur accelerated and optimized protocol bridges the advantages of AVITI sequencing with critical methods that rely on sequencing short fragments. This advance bolsters the utility of AVITI technology alongside traditional Illumina platforms, offering new opportunities for NGS applications.

Survey for Activating Oncogenic Mutation Variants in Metazoan Germline Genes.

Most cancers present with mutations or amplifications in distinctive tumor promoter genes that activate principal cell-signaling cascades promoting cell proliferation, dedifferentiation, cell survival, and replicative immortality. Somatic mutations found in this these driver proto-oncogenes invariably result in constitutive activation of the encoded protein. A salient feature of the activating mutations observed throughout many thousands of clinical tumor specimens reveals these driver missense mutations are recurrent and restricted to just one or very few codons of the entire gene, suggesting they have been positively selected during the course of tumor development. The purpose of this study is to investigate whether these characteristic oncogenic driver mutations are observed in the germline genes of any metazoan species. Six well-known tumor promoter genes were chosen for this survey including BRAF, KRAS, JAK2, PIK3CA, EGFR, and IDH1/2. The sites of all driver mutations were found to occur in highly conserved regions of each gene comparing protein sequences throughout diverse phyla of metazoan species. None of the oncogenic missense mutations were found in germlines of any species of current genome and protein databases. Despite many tumors readily selecting these somatic mutations, the conclusion drawn from this study is that these variants are negatively rejected if encountered as a germline mutation. While cancer expansion ensues from dysregulated growth elicited by these mutations, this effect is likely detrimental to embryonic development and/or survival of multicellular organisms. Although all oncogenic mutations considered here are gain-of-function where five of the six increase activity of the encoded proteins, clonal advancement promotes tumor growth by these genomic changes without conferring selection advantages benefiting the organism or species.

Pangenome Reveals Gene Content Variations and Structural Variants Contributing to Pig Characteristics.

Pigs are among the most essential sources of high-quality protein in human diets. Structural variants (SVs) are a major source of genetic variants associated with diverse traits and evolutionary events. However, the current linear reference genome of pigs limits the presentation of position information for SVs. In this study, we generated a pangenome of pigs and a genome variation map of 599 deep-sequenced genomes across Eurasia. Moreover, a section-wide gene repertoire was constructed, which indicated that core genes were more evolutionarily conserved than variable genes. Subsequently, we identified 546,137 SVs, their enrichment regions, and relationships with genomic features and found significant divergence across Eurasian pigs. More importantly, the pangenome-detected SVs could complement heritability estimates and genome-wide association studies based only on single nucleotide polymorphisms. Among the SVs shaped by selection, we identified an insertion in the promoter region of the TBX19 gene, which may be related to the development, growth, and timidity traits of Asian pigs and may affect the gene expression. Our constructed pig pangenome and the identified SVs provide rich resources for future functional genomic research on pigs.

Addressing pandemic-wide systematic errors in the SARS-CoV-2 phylogeny.

The SARS-CoV-2 genome occupies a unique place in infection biology - it is the most highly sequenced genome on earth (making up over 20% of public sequencing datasets) with fine scale information on sampling date and geography, and has been subject to unprecedented intense analysis. As a result, these phylogenetic data are an incredibly valuable resource for science and public health. However, the vast majority of the data was sequenced by tiling amplicons across the full genome, with amplicon schemes that changed over the pandemic as mutations in the viral genome interacted with primer binding sites. In combination with the disparate set of genome assembly workflows and lack of consistent quality control (QC) processes, the current genomes have many systematic errors that have evolved with the virus and amplicon schemes. These errors have significant impacts on the phylogeny, and therefore over the last few years, many thousands of hours of researchers time has been spent in "eyeballing" trees, looking for artefacts, and then patching the tree. Given the huge value of this dataset, we therefore set out to reprocess the complete set of public raw sequence data in a rigorous amplicon-aware manner, and build a cleaner phylogeny. Here we provide a global tree of 4,471,579 samples, built from a consistently assembled set of high quality consensus sequences from all available public data as of June 2024, viewable at https://viridian.taxonium.org . Each genome was constructed using a novel assembly tool called Viridian ( https://github.com/iqbal-lab-org/viridian ), developed specifically to process amplicon sequence data, eliminating artefactual errors and mask the genome at low quality positions. We provide simulation and empirical validation of the methodology, and quantify the improvement in the phylogeny. We hope the tree, consensus sequences and Viridian will be a valuable resource for researchers.

ChromaFold predicts the 3D contact map from single-cell chromatin accessibility

Identifying cell-type-specific 3D chromatin interactions between regulatory elements can help decipher gene regulation and interpret disease-associated non-coding variants. However, achieving this resolution with current 3D genomics technologies is often infeasible given limited input cell numbers. We therefore present ChromaFold, a deep learning model that predicts 3D contact maps, including regulatory interactions, from single-cell ATAC sequencing (scATAC-seq) data alone. ChromaFold uses pseudobulk chromatin accessibility, co-accessibility across metacells, and a CTCF motif track as inputs and employs a lightweight architecture to train on standard GPUs. Trained on paired scATAC-seq and Hi-C data in human samples, ChromaFold accurately predicts the 3D contact map and peak-level interactions across diverse human and mouse test cell types. Compared to leading contact map prediction models that use ATAC-seq and CTCF ChIP-seq, ChromaFold achieves state-of-the-art performance using only scATAC-seq. Finally, fine-tuning ChromaFold on paired scATAC-seq and Hi-C in a complex tissue enables deconvolution of chromatin interactions across cell subpopulations.

Optimization of PCA Error Correction Conditions to Improve Efficiency of Virus Genome De Novo Synthesis.

In recent years, there have been frequent global outbreaks of viral epidemics such as Zika, COVID-19, and monkeypox, which have had a huge impact on human health and society and have also spurred innovation in virus engineering technology. The rise of synthetic virus genome technology has provided researchers with a new platform to accelerate vaccine and drug development. Although DNA synthesis technology has made significant progress, the current virus genome synthesis technology still requires the assembly of short oligonucleotides of around 60 bp into kb-level lengths when constructing long segments, a process in which the commonly used polymerase chain reaction assembly (PCA) technology has high error rates and is cumbersome to operate. This study optimized the error correction conditions after PCA assembly, increasing the accuracy of synthesizing 1 kb DNA fragments from 4.2 ± 2.1% before error correction to 31.3 ± 3.1% after two rounds of correction, an improvement of over 6 times. This study provides a more efficient operational process for synthesizing virus genomes from scratch, indicating greater potential for virus engineering in epidemic prevention and control and the field of biomedicine.

Leveraging Synthetic Virology for the Rapid Engineering of Vesicular Stomatitis Virus (VSV)

Vesicular stomatitis virus (VSV) is a prototype RNA virus that has been instrumental in advancing our understanding of viral molecular biology and has applications in vaccine development, cancer therapy, antiviral screening, and more. Current VSV genome plasmids for purchase or contract virus services provide limited options for modification, restricted to predefined cloning sites and insert locations. Improved methods and tools to engineer VSV will unlock further insights into long-standing virology questions and new opportunities for innovative therapies. Here, we report the design and construction of a full-length VSV genome. The 11,161 base pair synthetic VSV (synVSV) was assembled from four modularized DNA fragments. Following rescue and titration, phenotypic analysis showed no significant differences between natural and synthetic viruses. To demonstrate the utility of a synthetic virology platform, we then engineered VSV with a foreign glycoprotein, a common use case for studying viral entry and developing anti-virals. To show the freedom of design afforded by this platform, we then modified the genome of VSV by rearranging the gene order, switching the positions of VSV-P and VSV-M genes. This work represents a significant technical advance, providing a flexible, cost-efficient platform for the rapid construction of VSV genomes, facilitating the development of innovative therapies.

Efficient indexing and querying of annotations in a pangenome graph.

The current reference genome is the backbone of diverse and rich annotations. Simple text formats, like VCF or BED, have been widely adopted and helped the critical exchange of genomic information. There is a dire need for tools and formats enabling pangenomic annotation to facilitate such enrichment of pangenomic references. The Graph Alignment Format (GAF) is a text format, tab-delimited like BED/VCF files, which was proposed to represent alignments. GAF could also be used to store paths representing annotations in a pangenome graph, but there are no tools to index and query them efficiently. Here, we present extensions to vg and HTSlib that provide efficient sorting, indexing, and querying for GAF files. With this approach, annotations overlapping a subgraph can be extracted quickly. Paths are sorted based on the IDs of traversed nodes, compressed with BGZIP, and indexed with HTSlib/tabix via our extensions for the GAF format. Compared to the binary GAM format, GAF files are easier to edit or inspect because they are plain text, and we show that they are twice as fast to sort and half as large on disk. In addition, we updated vg annotate, which takes BED or GFF3 annotation files relative to linear sequences and projects them into the pangenome. It can now produce GAF files representing these annotations' paths through the pangenome. We showcase these new tools on several applications. We projected annotations for all Human Pangenome Reference Consortium Year 1 haplotypes, including genes, segmental duplications, tandem repeats and repeats annotations, into the Minigraph-Cactus pangenome (GRCh38-based v1.1). We also projected known variants from the GWAS Catalog and expression QTLs from the GTEx project into the pangenome. Finally, we reanalyzed ATAC-seq data from ENCODE to demonstrate what a coverage track could look like in a pangenome graph. These rich annotations can be quickly queried with vg and visualized using existing tools like the Sequence Tube Map or Bandage.

Two high quality chromosome-scale genome assemblies of female and male silver pomfret (Pampus argenteus)

Pampus argenteus is a highly commercial marine fish whose population is declining sharply. Here, we generated a female P. argenteus genome, spanning 536.33 Mb with contig N50 of 1.79 Mb; 24070 genes (99.50% of 24,182) were functionally annotated. To improve quality of it, we assembled a 553.79 Mb genome of male fish with contig N50 of 24.75 Mb through HiFi and ultra-long ONT sequence technologies; 550.82 Mb were anchored onto 24 gap-free chromosomes; 22,892 genes (98.1% of 23,346) were functionally annotated; the QV value was 51.55 with 98.9% of BUSCO and 99.39% coverage of Illumina reads. Finally, we compared this genome with previous published one, revealing 37,301 SVs. 52.82 Mb and 18.05 Mb SDs were characterized in our and published assemblies, respectively, and 48.96 Mb PURs were constructed. Thus, this genome assembly exhibits excellent completeness, continuity and accuracy comparing to the published one, which can be current preferred reference genome. Overall, these works help aquaculture and wild resources recovery of P. argenteus and provide a valuable genetic resource for study.

The complete chloroplast genome of Tabebuia rosea (Bignoniaceae)

Tabebuia rosea is a world-renowned woody plant with colorful flowers in full bloom. In addition to its high ornamental value, it also has ecological and medicinal value. In this study, the complete circular chloroplast genome of T. rosea was reconstructed and annotated using Illumina sequencing. The chloroplast genome was 158,919 bp in size with GC content of 38.21%, including a large single-copy region of 85,823 bp, a small single-copy region of 12,816 bp, and a pair of inverted repeats of 30,140 bp. It encoded 132 genes, including 87 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Based on current available chloroplast genome sequences, the phylogenetic analysis indicated that T. rosea was clustered with T. nodosa and H. chrysanthus. This study provided insights into the evolutionary relationships among different species of Bignoniaceae.

Complete analysis of G-quadruplex forming sequences in the gapless assembly of human chromosome Y

Recent advancements have finally delivered a complete human genome assembly, including the elusive Y chromosome. This accomplishment closes a significant knowledge gap. Prior efforts were hampered by challenges in sequencing repetitive DNA structures such as direct and inverted repeats. We used the G4Hunter algorithm to analyze the presence of G-quadruplex forming sequences (G4s) within the current human reference genome (GRCh38) and the new telomere-to-telomere (T2T) Y chromosome assemblies. This analysis served a dual purpose: identifying the location of potential G4s within the genomes and exploring their association with functionally annotated sequences. Compared to GRCh38, the T2T assembly exhibited a significantly higher prevalence of G-quadruplex forming sequences. Notably, these repeats were abundantly located around precursor RNA, exons, genes, and within protein binding sites. This remarkable co-occurrence of G4-forming sequences with these critical regulatory regions suggests their role in fundamental DNA regulation processes. Our findings indicate that the current human reference genome significantly underestimated the number of G4s, potentially overlooking their functional importance.

Improved draft genome of Ignatzschineria larvae DSM 13226 via hybrid assembly.

Ignatzschineria larvae is studied for its role in decomposition and disease ecology; however, the type strain reference genome remains fragmented. The current reference genome consists of 61 contigs calculated at 82.18% complete with 10.98% contamination. Here, we announce the hybrid genome assembly as an improved single contig.

A novel role of the cotton calcium sensor CBL3 was involved in Verticillium wilt resistance in cotton.

Verticillium wilt, causes mainly by the soilborne pathogen Verticillium dahliae, is a devastated vascular disease resulting in huge financial losses in cotton, so research on improving V. dahliae stress tolerance in cotton is the utmost importance. Calcium as the second messenger acts as a crucial role in plant innate immunity. Cytosolic Ca2+during the pathogen infection is a significant increase in plant immune responses. Calcineurin B-like (CBL) proteins are widely known calcium sensors that regulate abiotic stress responses. However, the role of cotton CBLs in response to V. dahliae stress remains unclear. To discover and utilize the gene to Verticillium wilt resistance and defense response mechanism of cotton. Through screening the gene to Verticillium wilt resistance in cotton, four GhCBL3 copies were obtained from the current common cotton genome sequences. The protein domain and phylogenetic analyses of GhCBL3 were performed using NCBI Blast, DNAMAN, and MotifScan programs. Real-time RT-PCR was used to detect the expression of GhCBL3 gene in cotton seedlings under various stress treatments. The expression construct including GhCBL3 cDNA was transduced into Agrobacterium tumefaciens (GV3101) by heat shock method and transformed into cotton plants by Virus-Induced Gene Silencing (VIGS) method. The results of silencing of GhCBl3 on ROS accumulation and plant disease resistance in cotton plants were assessed. A member of calcineurin B-like proteins (defined as GhCBL3) in cotton was obtained. The expression of GhCBL3 was significantly induced and raised by various stressors, including dahliae, jasmonic acid (JA) and H2O2 stresses. Knockdown GhCBL3 in cotton by Virus-Induced Gene Silencing analysis enhanced Verticillium wilt tolerance and changed the occurrence of reactive oxygen species. Some disease-resistant genes were increased in GhCBL3-silencing cotton lines. GhCBL3 may function on regulating the Verticillium dahliae stress response of plants.

Refinement of the Antarctic fur seal (Arctocephalus gazella) reference genome increases continuity and completeness.

The Antarctic fur seal (Arctocephalus gazella) is an important top predator and indicator of the health of the Southern Ocean ecosystem. Although abundant, this species narrowly escaped extinction due to historical sealing and is currently declining as a consequence of climate change. Genomic tools are essential for understanding these anthropogenic impacts and for predicting long-term viability. However, the current reference genome ("arcGaz3") shows considerable room for improvement in terms of both completeness and contiguity. We therefore combined PacBio sequencing, haplotype-aware HiRise assembly, and scaffolding based on Hi-C information to generate a refined assembly of the Antarctic fur seal reference genome ("arcGaz4_h1"). The new assembly is 2.53 Gb long, has a scaffold N50 of 55.6 Mb and includes 18 chromosome-sized scaffolds, which correspond to the 18 chromosomes expected in otariids. Genome completeness is greatly improved, with 23,408 annotated genes and a Benchmarking Universal Single-Copy Orthologs score raised from 84.7% to 95.2%. We furthermore included the new genome in a reference-free alignment of the genomes of 11 pinniped species to characterize evolutionary conservation across the Pinnipedia using genome-wide Genomic Evolutionary Rate Profiling. We then implemented Gene Ontology enrichment analyses to identify biological processes associated with those genes showing the highest levels of either conservation or differentiation between the 2 major pinniped families, the Otariidae and Phocidae. We show that processes linked to neuronal development, the circulatory system, and osmoregulation are overrepresented both in conserved as well as in differentiated regions of the genome.

Plant Disease: A Growing Threat to Global Food Security

The escalating global population has led to an increased demand for both quantity and quality in food production. Throughout history, plant diseases have posed significant threats to agricultural output by causing substantial food losses annually while also compromising product quality. Accurate identification of pathogens, clarifying the pathogenic mechanism of pathogens, and understanding the interaction between pathogens and hosts are important for the control of plant diseases. This Special Issue, “Research Progress on Pathogenicity of Fungi in Crops”, belongs to the section “Pest and Disease Management” of Agronomy. It contains research papers on the identification and phylogeny of fungal pathogens, the molecular genetics of plant fungal pathogens, the molecular mechanisms of fungal pathogenicity, and the molecular basis of the interaction between fungi and crops. These studies encapsulate efforts to understand disease systems within current genomics, transcriptomics, proteomics, and metabolomics studies, highlighting research findings that could be future targets for crop disease and pest control. The studies presented in this Special Issue promote the progress of fungal pathogenicity research in crops and provide a scientific basis for future disease control, which is of great significance for sustainable agricultural development and global food security.

Chromosome-level genome assembly of Plagiognathops microlepis based on PacBio HiFi and Hi-C sequencing

Plagiognathops microlepis is an economic freshwater fish in the subfamily Xenocyprinae of Cyprinidae. It is widely distributed in the freshwater ecosystem of China, with moderate economic value and broad development prospects. However, the lack of genomic resources has limited our understanding on the genetic basis, phylogenetic status and adaptive evolution strategies of this fish. Here, we assembled a chromosome-level reference genome of P. microlepis by integrating Pacbio HiFi long-reads, Illumina short-reads and Hi-C sequencing data. The size of this genome is 1004.34 Mb with a contig N50 of 38.80 Mb. Using Hi-C sequencing data, 99.59% of the assembled sequences were further anchored to 24 chromosomes. A total of 578.91 Mb repeat sequences and 28,337 protein-coding genes were predicted in the current genome, of which, 26,929 genes were functionally annotated. This genome provides valuable information for investigating the phylogeny and evolutionary history of cyprinid fishes, as well as the genetic basis of adaptive strategies and special traits in P. microlepis.

EquCab_Finn: A new reference genome assembly for the domestic horse, Finnhorse.

Finnhorse is Finland's native and national horse breed and it has genetic affinities to northern European and Asian horses. It has historical importance for agriculture, forest work and transport and as a war horse. Finnhorse has four breeding sections in the studbook and is under conservation and characterisation efforts. We sequenced and annotated the genome of a Finnhorse mare from the working horse section using PacBio and Omni-C data. This genome can complement the existing Thoroughbred reference genome (EquCab 3.0) and facilitate genetic studies of horses from northern Eurasia. We assembled 2.4 Gb of the genome with an N50 scaffold length of 83.8 Mb and the genome annotation resulted in a total of 19 748 protein coding genes of which 1200 were Finnhorse specific. The assembly has high quality and synteny with the current horse reference genome. We manually curated five genes of interest and deposited the final assembly in the European Nucleotide Archive under the accession no. PRJEB71364.

Improved assembly of the Pungitius pungitius reference genome.

The nine-spined stickleback (Pungitius pungitius) has been increasingly used as a model system in studies of local adaptation and sex chromosome evolution but its current reference genome assembly is far from perfect, lacking distinct sex chromosomes. We generated an improved assembly of the nine-spined stickleback reference genome (98.3% BUSCO completeness) with the aid of linked-read mapping. While the new assembly (v8) was of similar size as the earlier version (v7), we were able to assign 4.4 times more contigs to the linkage groups and improve the contiguity of the genome. Moreover, the new assembly contains a ∼22.8 Mb Y-linked scaffold (LG22) consisting mainly of previously assigned X-contigs, putative Y-contigs, putative centromere contigs, and highly repetitive elements. The male individual showed an even mapping depth on LG12 (pseudo X chromosome) and LG22 (Y-linked scaffold) in the segregating sites, suggesting near-pure X and Y representation in the v8 assembly. A total of 26,803 genes were annotated, and about 33% of the assembly was found to consist of repetitive elements. The high proportion of repetitive elements in LG22 (53.10%) suggests it can be difficult to assemble the complete sequence of the species' Y chromosome. Nevertheless, the new assembly is a significant improvement over the previous version and should provide a valuable resource for genomic studies of stickleback fishes.