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PubMed2026

ImpuT2T: Pangenome-Based Patching for Human Genome Assemblies.

With improvements in sequencing and assembly have come many high-quality telomere-to-telomere assemblies and reference pangenomes. However, the long-read sequencing recipes needed for high quality assemblies are expensive, and out of reach for many research groups. Here we propose ImpuT2T, a method that takes an assembly produced via inexpensive HiFi sequencing reads, and uses a panel of T2T (or near-T2T) assemblies to scaffold and fill ("patch") the gaps between the HiFi contigs. Benchmarking against reference assemblies demonstrates that ImpuT2T is highly effective at patching human HiFi assemblies, consistently outperforming existing patching approaches. Moreover, we show that including more haplotypes in the pangenome improves the quality of the patched assemblies, with the greatest gains achieved using the full HPRC Release 2 pangenome.

Journal Article↗

PubMed2025

A chromosome-level genome assembly of Guimi No. 2 (Actinidia chinensis).

In this study, we report a high-quality chromosome-level genome assembly of Actinidia chinensis var. chinensis 'Guimi No. 2'. This cultivar, discovered in Guizhou karst ecosystems, exhibits resistance to Pseudomonas syringae pv. actinidiae (Psa). Using a combination of MGI short-read sequencing, PacBio HiFi long-read sequencing, and Hi-C technology, we generated a genome assembly of 608.43 Mb with a contig N50 of 20.70 Mb, and 99.70% of the assembly was successfully anchored onto 29 pseudochromosomes. The quality value (QV) and the LTR Assembly Index (LAI) of the assembled genome were 72.23 and 10.10. The BUSCO analysis indicated that the genome assembly and gene model prediction were 98.40% and 96.56% complete, respectively. A total of 251.15 Mb of repetitive sequences and 45,986 protein-coding genes were annotated. This genome assembly provides critical insights into A. chinensis's genomic architecture and serves as a foundational resource for elucidating disease resistance mechanisms against Psa, while enabling comparative phylogenomic studies across the Actinidia genus.

Actinidia↗

PubMed2026

Amaranth: enhanced single-cell transcript assembly via discriminative modelling of UMI reads and internal reads.

MOTIVATION: Single-cell RNA sequencing (scRNA-seq) has transformed transcriptome profiling at cellular resolution, yet accurate reconstruction of full-length transcripts for individual cells remains a central challenge. Emerging scRNA-seq protocols can produce reads that span entire transcripts, enabling isoform-level expression analysis. For example, Smart-seq protocols combine unique molecular identifier (UMI)-linked reads that index and stitch together multiple reads from the same molecule, with internal reads filling coverage gaps. We demonstrate that these read types exhibit markedly different biological and statistical properties in strandness, 5'/3' coverage bias, and genomic locality. Existing assemblers fail to leverage these distinctions, yielding suboptimal assembly. RESULTS: We developed Amaranth, a novel single-cell assembler that discriminatively models UMI and internal reads. Amaranth implements heuristics specifically designed to address the distinct biases of UMI-linked and internal reads, enabling accurate strandness assignment for internal reads, reliable splicing graph refinement, and precise transcript start site determination. We also developed Amaranth-meta, which integrates information across cells to enhance individual cell assemblies. Benchmarked on Smart-seq3 datasets from human HEK293T and mouse fibroblast cells, Amaranth outperformed other state-of-the-art assemblers in assembling individual cells and in meta-assembly. Amaranth advances isoform-level analysis in single-cell transcriptomics, facilitating detailed studies at cellular resolution. AVAILABILITY AND IMPLEMENTATION: Amaranth is implemented in C++ and is freely available at https://github.com/Shao-Group/amaranth under the BSD-3-Clause license. Scripts, documentation, and data for reproducing experiments in this manuscript are available at https://github.com/Shao-Group/amaranth-test.

Single-Cell Gene Expression Analysis↗

PubMed2025

High-resolution metagenome assembly for modern long reads with myloasm.

Long-read metagenome assembly promises complete genomic recovery from microbiomes. However, the complexity of metagenomes poses challenges. We present myloasm, a metagenome assembler for PacBio HiFi and Oxford Nanopore Technologies (ONT) R10.4 long reads. Myloasm uses polymorphic k-mers to construct a high-resolution string graph and then leverages differential abundance for graph simplification. On real-world ONT metagenomes, myloasm assembled three times more complete circular contigs than the next-best assembler. Myloasm can make ONT and HiFi comparable for assembly: for a jointly sequenced gut metagenome, myloasm with ONT assembled more complete circular genomes than any assembler with HiFi. Myloasm recovers previously inaccessible within-species diversity; we recovered six complete Prevotella copri single-contig genomes from a gut metagenome and eight complete TM7 (Saccharibacteria) contigs with > 93% similarity from an oral metagenome. With this improved resolution, we resolved two 98% similar ermF antibiotic resistance genes spreading through distinct strain-specific mobile genetic elements in a human gut.

Journal Article↗

PubMed1978

Stoichiometry and role of GTP hydrolysis in bovine neurotubule assembly.

A method is given for preparing tubulin with 1 mol of exchangeably bound [gamma-32P]GTP/mol of 6 S dimer. Bovine tubulin is shown to hydrolyze 1 mol of GTP/mol of 6 S dimer added to assembling microtubules at 37 degrees. Hydrolysis and assembly occur at the same rate and to the same extent. When microtubule-associated proteins (MAPs) are removed, both hydrolysis and assembly fail to occur. Readdition of the MAPs restores both activities. Tubulin with exchangeable GDP will co-assemble with GTP.tubulin even at equimolar levels. Exchangeability is demonstrated by pulse-chase experiments with GDP or GTP. GDP is also a potent inhibitor of assembly under these conditions, and the rate of assembly is reduced by 50% at 10 micron GDP. One mole of inorganic phosphate is released to the solvent per mole of exchangeable GTP hydrolyzed. An assembly mechanism is proposed in which exchangeable GTP is hydrolyzed without intermediate transphosphorylation of nonexchangeable GDP.

Animals↗

PubMed2026

Characterization of a draft chromosome-scale genome assembly for the mutton snapper, Lutjanus analis.

BACKGROUND: The mutton snapper (Lutjanus analis) is a reef fish commonly found in tropical waters of the Western Atlantic Ocean. Genomic studies of this species are needed to support conservation efforts and breeding programs. OBJECTIVE: Here, we report the development of a chromosome-scale reference assembly for the mutton snapper and conduct an initial comparative genomic analysis with other lutjanids. METHODS: The genome of one mutton snapper specimen was sequenced using PAC-Bio HiFi long reads and Illumina short reads. Contigs and scaffolds were assembled in the Flye pipeline and anchored using Hi-C proximity guided assembly. Gene prediction and functional annotations were obtained in AUGUSTUS and eggNOG-mapper, respectively. The mutton snapper genome was compared to those of other lutjanids to infer gene family evolution and chromosome synteny conservation. RESULTS: Assembly and polishing yielded 946 contigs and 926 scaffolds (N50 of 3.16 Mb, complete BUSCO score 98.1%) that were anchored using Hi-C scaffolding in 24 draft chromosomes. The anchored assembly featured a N50 of 42.47 Mb and contained 97.6% of the unanchored assembly length. The 24 mutton snapper chromosomes showed a one-to-one syntenic relationship with their counterparts in medaka, and other Lutjanids. AUGUSTUS predicted 29,023 genes, 24,335 of which (83.85%) could be functionally annotated. Gene family evolution analysis revealed 1,014 significantly expanded or contracted hierarchical ortholog groups in mutton snapper. Expansions and contractions were linked to several biological functions including growth, oocyte maturation, and response to exogenous stressors. CONCLUSION: The draft genome will be a valuable tool for forthcoming applied genomic studies of mutton snapper.

Animals↗

PubMed1977

Assembly of SV40 chromatin in a cell-free system from Xenopus eggs.

A cell-free system is described which assembles chromatin from purified DNA in 1 hr under physiological incubation conditions. It consists of a 145,000 x g (maximum) supernatant fraction from eggs of Xenopus laevis. It converts SV40 DNA to a nucleoprotein which co-sediments with naturally occurring SV40 chromatin and which can be cleaved by micrococcal nuclease to a highly ordered pattern of DNA fragments resembling those from digestion of liver chromatin. It inserts superhelical turns into relaxed, covalently closed DNA. The assembly process is not cooperative. Under limiting conditions, each DNA molecule becomes partially assembled. Assembly does not require replication of the DNA or protein synthesis, but occurs from a stored histone pool of at least 40 ng per egg. Under conditions of DNA excess, assembly becomes dependent upon the amount of exogenous histones added to the incubation. Apart from histones and a nicking-closing activity, chromatin assembly requires an additonal thermolabile factor which is present in the egg supernatant.

Animals↗

PubMed1978

Tubulin assembly protein: immunochemical and immunofluorescent studies on its function and distribution in microtubules and cultured cells.

Cytoplasmic microtubule assembly from tubulin monomers requires an accessory protein or proteins present is isolated microtubules. These proteins have been designated "tau" factors. One such factor, tubulin assembly protein (TAP), has been purified to homogeneity from calf brain microtubules. A precipitating, monospecific antibody against the protein has been prepared. The antibody has been used to investigate the mechanism of TAP action in microtubule assembly and the distribution of TAP in cellular microtubules. Immunochemical, immunofluorescent and electron microscopic studies indicate that TAP functions stoichiometrically by binding physically to tubulin to form a complex active in microtubule assembly. TAP is an elongation protein which is required throughout the growth of a microtubule and which is actually present along the entire microtubule. Immunofluorescence microscopy has been used to demonstrate that TAP is distributed throughout the cytoplasmic microtubule network of cultured human, hamster and rat cells-both normal and virally transformed. Immunofluorescence of cells in mitosis shows that TAP is present in the mitotic spindle. These results demonstrate the biological importance of tubulin assembly protein and suggest that it or immunologically related "tau" proteins represent ubiquitous cofactors in cytoplasmic microtubule assembly.

Antibodies↗

PubMed2025

Chromosome-level genome assembly and annotation of Pterygoplichthys pardalis.

Suckermouth catfishes, with their evolved powerful features, have become notorious invasive species, causing significant damage to aquatic ecosystems. However, the lack of high-quality genomes severely restricts research on this group within the field. In this study, we de novo assembled the chromosome-level genome assembly of Pterygoplichthys pardalis using multiple platforms of sequencing data, including Illumina short reads, Nanopore long reads, and Hi-C sequencing reads, resulting in a 1.51 Gb genome assembly. Multiple evaluations, including read mapping ratio (98.52%), transcript mapping ratio (99.61%), conserved BUSCO gene set (98.8%), and N50 score (49.47 Mb), indicated the high continuity and accuracy of the genome assembly we generated. Genome annotation found that 0.97 Gb of genome sequences are repetitive sequences, accounting for 64.47% of the genome assembly. Further, 23,859 protein-coding genes were successfully predicted, 92.92% of which could be annotated in functional databases. This high-quality genome assembly of P. pardalis provides a valuable resource for understanding the genetic underpinnings of P. pardalis's invasive success and offers critical data for future fisheries research and management.

Animals↗

PubMed2025

Chromosome-level genome assembly of Sinocyclocheilus jii based on PacBio HiFi and Hi-C sequencing.

Sinocyclocheilus jii, a cavefish species endemic to China, belongs to the genus Sinocyclocheilus within the family Cyprinidae. Species within this genus exhibit significant morphological differentiation, making it not only the most species-rich genus within Cyprinidae in China but also the most diverse group of cavefishes worldwide. However, the limited availability of genomic resources has limited investigations into the genetic basis of trait variations, phylogenetic relationships, and adaptive evolution in this genus. In this study, we assembled a chromosome-level reference genome for S. jii by integrating PacBio HiFi long reads, Illumina short reads, and Hi-C sequencing data. Flow cytometry was used to estimate the genome size prior to assembly, providing a key step in technical validation. The final genome assembly spans 1.75 Gb with a contig N50 of 35.0 Mb. Using Hi-C sequencing data, the assembled scaffolds were successfully anchored to 50 chromosomes. The completeness of the chromosome-level assembly was estimated at 98.9% by BUSCO analysis. Genome annotation identified 855.5 Mb of repetitive sequences and predicted a total of 52,867 protein-coding genes, of which 51,932 genes were functionally annotated. This study presents a high-quality chromosome-level genome assembly and annotation of S. jii, providing a fundamental genomic resource for future phylogenetic and evolutionary studies.

Animals↗

PubMed2026

High quality genome assemblies of African cattle breeds using PacBio HiFi sequencing.

Africa has a uniquely rich cattle diversity of ~150 breeds comprising the Bos taurus indicus sub-species, Bos taurus taurus, and their crosses. These represent ~23% of the global cattle population. However, high quality, representative assemblies are limited for African cattle and especially for indicine breeds. Here we built high quality de novo assemblies for five important African indigenous cattle breeds using PacBio HiFi sequencing: Lagune (Bos taurus taurus), Gudali, Iringa Red and Singida White (Bos taurus indicus), and Mpwapwa (Bos taurus taurus x Bos taurus indicus). These new assemblies are the most contiguous and complete African cattle assemblies produced so far, with genome sizes of 3.25-3.36 Gb, contiguity N50s ranging from 83.59 Mb to 97.87 Mb and scaffold N50s from 100.30 Mb to 113.37 Mb. BUSCO genome completeness scores were also higher than 99.68%, indicative of highly contiguous assemblies. These improved and highly contiguous genome assemblies are consequently a valuable resource for future African and global livestock genomic studies.

Animals↗

PubMed2026

Multidomain interaction governs the filamentous assembly of the dominant-negative DNMT3A R882H mutant.

DNA methyltransferase DNMT3A-mediated de novo DNA methylation is important for proper regulation of gene expression and genomic stability in development. The DNMT3A R882H (DNMT3AR882H) mutation, a hot-spot mutation in acute myeloid leukemia and developmental disorders, exerts a dominant-negative effect in DNMT3A-mediated DNA methylation through promoting high-order protein assembly. However, due to the lack of structural knowledge on DNMT3A homo-oligomers, the mechanism behind wild-type DNMT3A (DNMT3AWT) and DNMT3AR882H polymerization remains unclear. Here, we report the single-particle cryo-EM structures of homo-oligomeric DNMT3AWT and filamentous DNMT3AR882H, revealing the role of the regulatory Pro-Trp-Trp-Pro (PWWP) and ATRX-DNMT3-DNMT3L (ADD) domains of DNMT3A in their dynamic assembly. While the oligomeric assembly of DNMT3A is mainly driven by the well-characterized oligomer interfaces in the methyltransferase domain, the autoinhibitory interaction of the PWWP and ADD domains in DNMT3A places them in a position for intermolecular contact, thereby contributing to the filamentous assembly of DNMT3AR882H. Disrupting the autoinhibitory interaction facilitates the transition of DNMT3AR882H polymer toward the low-order oligomeric assembly, reinforcing the aggregation-attenuation effect of the previously characterized oligomer-interface mutation R676K. Together, this study uncovers a multidomain cooperated assembly mechanism for DNMT3A, with important implication in development of effective therapeutic strategies against DNMT3AR882H-associated diseases.

DNA (Cytosine-5-)-Methyltransferases↗

PubMed1977

Membrane-associated assembly of M13 phage in extracts of virus-infected Escherichia coli.

Assembly of coliphage M13 is known to occur as the viral DNA crosses the cytoplasmic membrane, shedding its virus-coded DNA unwinding protein and acquiring from the membrane approximately 2400 copies of the major coat protein. Conditions are described in which extracts of M13-infected E. coli and membranes prepared from such extracts will support virus assembly at a rate equivalent to that of intact cells. Extracts prepared from cells infected with temperature-sensitive M13 mutants in genes 1, 3, 4, or 5 are temperature-sensitive in this cell-free assembly reaction. Phage assembly in vitro requires magnesium and as yet an unidentified heat-stable cofactor of low molecular weight. The rate of virus assembly is approximately linear with respect to extract concentration over a 10(4)-fold range, consistent with the observation that the entire M13 assembly activity copurifies with the cell membrane fraction.

Cell Membrane↗

PubMed1977

Role of surface modulating assemblies in growth control of normal and transformed fibroblasts.

Cellular microtubules, microfilaments, and surface receptors have been postulated to form a surface modulating assembly that regulates surface receptor mobility and cell growth. To test this hypothesis, we examined three agents known to affect cell growth [colchicine, concanavalin A (Con A), and the src gene product of Rous sarcoma virus] for their effects on chick embryo fibroblasts. Individual cells from serum-starved normal fibroblast populations became committed to enter S phase at various times over a 12 hr period after exposure to serum. Colchicine and other microtubule-disrupting agents blocked entry into S phase at a point close to the commitment point for each cell. The lectin Con A also blocked entry into the S phase when present in doses sufficient to modulate surface receptor mobility. In contrast, succinyl-Con A, which does not induce surface modulation, had no effect. Both Con A and colchicine blocked the appearance of cytoplasmic factors capable of stimulating DNA replication in a cell-free system. To study endogenous effects on the surface modulating assembly, we infected fibroblasts with a Rous sarcoma virus (tsNY68) having a temperature-sensitive mutation in the transforming (src) gene. We have previously shown that microtubular and microfilamentous structures of the surface modulating assembly are direct or indirect targets of the src gene product with consequent reduction in the capacity of Con A to induce surface modulation. TsNY68-infected fibroblasts shifted to the non-permissive temperature acquired normal microtubular morphology more rapidly (2 hr) than cells grown at the permissive temperature in the presence of protein synthesis inhibitors (7.5 hr). This suggests that the src gene product acts directly on the surface modulating assembly rather than via the nucleus or at the level of protein synthesis. Furthermore, "transformation" of the surface modulating assembly was partly blocked by treatment of the infected cells with Con A but not succinyl-Con A. Both Con A and colchicine inhibited entry into the S phase following a shift from nonpermissive to permissive growth conditions. All of these observations are in accord with the hypothesis that the surface modulating assembly acts as a signal regulator in growth control.

Animals↗

PubMed1979

Cell cycle-dependent, in vitro assembly of microtubules onto pericentriolar material of HeLa cells.

A centriolar complex comprising a pair of centrioles and a cloud of pericentriolar materials is located at the point of covergence of the microtubules of the mitotic apparatus. The in vitro assembly of microtubules was observed onto these complexes in the 1,400 g supernatant fraction of colcemid-blocked, mitotic HeLa cells lysed into solutions containing tubulin and Triton X-100. Dark-field microscopy provided a convenient means by which this process could be visualized directly. When this 1,400 g supernate was incubated at 30 degrees C and centrifuged into a discontinuous sucrose gradient, a band containing centriolar complexes and assembled microtubles was obtained at 50-60% sucrose interface. Ultrastructual analysis indicated that the majority of the microtubules assembled predominantly from the pericentriolar material but also onto the centrioles. When cells were synchronized by a double thymide block, the assembly of microtubules onto centriolar complexes was observed only in lysates of mitotic cells; no assembly was seen in lysed material of interphase cells. Microtubule assembly occured onto centriolar complexes in solutions of either 100,000 g brain supernate, 2 X cycled tubulin, or purified tubulin dimers. This study demonstrates that the pericentriolar material becomes competent as a microtubule-organizing center (MTOC) at the time of mitosis. With use of the techniques described, a method for the isolation of centriolar complexes may be developed.

Cell Cycle↗

PubMed2025

Mapler: a pipeline for assessing assembly quality in taxonomically rich metagenomes sequenced with HiFi reads.

SUMMARY: Metagenome assembly seeks to reconstruct the most high-quality genomes from sequencing data of microbial ecosystems. Despite technological advancements that facilitate assembly, such as Hi-Fi long reads, the process remains challenging in complex environmental samples consisting of hundreds to thousands of populations. Mapler is a metagenome assembly and evaluation pipeline with a focus on evaluating the quality of Hi-Fi long read metagenome assemblies. It incorporates several state-of-the-art metrics, as well as novel metrics assessing the diversity that remains uncaptured by the assembly process. Mapler facilitates the comparison of assembly strategies and helps identify methodological bottlenecks that hinder genome reconstruction. AVAILABILITY AND IMPLEMENTATION: Mapler is open source and publicly available under the AGPL-3.0 licence at https://github.com/Nimauric/Mapler. Source code is implemented in Python and Bash as a Snakemake pipeline. A snapshot of the code is available on Software Heritage at swh:1:snp:df4f5f02e22ebbab285ec14af58d4d88436ee5d6. Raw data and results are available at https://entrepot.recherche.data.gouv.fr/dataset.xhtml?persistentId=doi:10.57745/2SA8AB.

Metagenome↗

PubMed2026

De novo assembly and authentication of ancient DNA metagenomes with nf-core/mag.

Ancient DNA provides a direct window into the evolutionary processes that have shaped living microbial species today, as well as their now extinct relatives. Advances in both sequencing methods and de novo assembly techniques have not only resulted in a flood of modern metagenomic sequencing data, but they have also allowed palaeogenomicists to retrieve vast amounts of ancient DNA from past microorganisms, including species and strains without modern reference genomes. However, the degraded nature of ancient DNA means that the standard techniques of genome assembly developed for modern DNA are unlikely to perform effectively, unless heavily modified. This hinders the incorporation of ancient data into broader metagenomic studies that would otherwise benefit from having deep time information on the evolution of different microbial species. In this primer and protocol paper, we provide guidance on ways to adapt existing metagenomic de novo assembly processes, including data input, tools, and settings, in order to perform more robustly and effectively on ancient DNA. After assembly, we then further describe how ancient DNA contigs can be identified and validated. The key steps of ancient metagenomic assembly are now integrated in a dedicated ancient DNA mode in the established pipeline nf-core/mag. By introducing support for ancient DNA data in nf-core/mag, we aim to improve the ability of researchers to more regularly integrate de novo assembled ancient microbial data into broader metagenomics studies of microbial ecology and evolution.

DNA, Ancient↗

PubMed2025

Modularization of the type II secretion gene cluster from Xanthomonas euvesicatoria facilitates the identification of a structurally conserved XpsCLM assembly platform complex.

Many bacterial pathogens depend on a type II secretion (T2S) system to secrete virulence factors from the periplasm into the extracellular milieu. T2S systems consist of an outer membrane secretin channel, a periplasmic pseudopilus and an inner membrane-associated assembly platform including a cytoplasmic ATPase. The components of T2S systems are often conserved in different bacterial species, however, the architecture of the assembly platform is largely unknown. Here, we analysed predicted assembly platform components of the Xps-T2S system from the plant-pathogenic bacterium Xanthomonas euvesicatoria. To facilitate these studies, we generated a modular xps-T2S gene cluster by Golden Gate assembly of single promoter and gene fragments. The modular design allowed the efficient deletion and replacement of T2S genes and the insertion of reporter fusions. Mutant approaches as well as interaction and crosslinking studies showed that the predicted assembly platform components XpsC, XpsL and XpsM form a trimeric complex which is essential for T2S and associates with the cytoplasmic ATPase XpsE and the secretin XpsD. Structural modeling revealed a similar trimeric architecture of XpsCLM homologs from Pseudomonas, Vibrio and Klebsiella species, despite overall low amino acid sequence similarities. In X. euvesicatoria, crosslinking and fluorescence microscopy studies showed that the formation of the XpsCLM complex is independent of the secretin and vice versa, suggesting that the assembly of the T2S system is a dynamic process which involves the association of preformed subcomplexes.

Xanthomonas↗