Complete Assembly Research Articles

Emission and performance characterization of ionic liquids for an externally wetted electrospray thruster

Ionic liquids are the base for electrospray propulsion, yet few have been explored. The performance of an externally wetted electrospray thruster is characterized for seven different ionic liquids: 1-ethyl-3-methylimidazolium tetrafluoroborate (EMI-BF4), dicyanamide (EMI-DCA), bistrifluoromethylsulfonylimide (EMI-Im), thiocyanate (EMI-SCN), ethylsulphate (EMI-EtOSO3), triflate (EMI-OTf), and trifluoromethanesulfonate (EMI-TFA). This research marks the first time that the latter four liquids are reported for externally wetted emitters. A complete thruster assembly has been tested with each of the propellants. Using time-of-flight mass spectrometry and a Faraday cup for beam divergence, the performance of these ionic liquids has been characterized, assessing thrust, specific impulse and thrust efficiency. EMI-BF4, EMI-DCA and EMI-SCN operated in pure ionic regime while EMI-Im, EMI-EtOSO3, EMI-OTf and EMI-TFA operated in mixed regime.

Constructing telomere-to-telomere diploid genome by polishing haploid nanopore-based assembly.

Draft genomes generated from Oxford Nanopore Technologies (ONT) long reads are known to have a higher error rate. Although existing genome polishers can enhance their quality, the error rate (including mismatches, indels and switching errors between paternal and maternal haplotypes) can be significant. Here, we develop two polishers, hypo-short and hypo-hybrid to address this issue. Hypo-short utilizes Illumina short reads to polish an ONT-based draft assembly, resulting in a high-quality assembly with low error rates and switching errors. Expanding on this, hypo-hybrid incorporates ONT long reads to further refine the assembly into a diploid representation. Leveraging on hypo-hybrid, we have created a diploid genome assembly pipeline called hypo-assembler. Hypo-assembler automates the generation of highly accurate, contiguous and nearly complete diploid assemblies using ONT long reads, Illumina short reads and optionally Hi-C reads. Notably, our solution even allows for the production of telomere-to-telomere diploid genomes with additional manual steps. As a proof of concept, we successfully assembled a fully phased telomere-to-telomere diploid genome of HG00733, achieving a quality value exceeding 50.

Complete genome assembly provides a high-quality skeleton for pan-NLRome construction in melon.

Melon (Cucumis melo L.), being under intensive domestication and selective breeding, displays an abundant phenotypic diversity. Wild germplasm with tolerance to stress represents an untapped genetic resource for discovery of disease-resistance genes. To comprehensively characterize resistance genes in melon, we generate a telomere-to-telomere (T2T) and gap-free genome of wild melon accession PI511890 (C. melo var. chito) with a total length of 375.0 Mb and a contig N50 of 31.24 Mb. The complete genome allows us to dissect genome architecture and identify resistance gene analogs. We construct a pan-NLRome using seven melon genomes, which include 208 variable and 18 core nucleotide-binding leucine-rich repeat receptors (NLRs). Multiple disease-related transcriptome analyses indicate that most up-regulated NLRs induced by pathogens are shell or cloud NLRs. The T2T gap-free assembly and the pan-NLRome not only serve as essential resources for genomic studies and molecular breeding of melon but also provide insights into the genome architecture and NLR diversity.

A pooled-sample draft genome assembly provides insights into host plant-specific transcriptional responses of a Solanaceae-specializing pest, Tupiocoris notatus (Hemiptera: Miridae).

The assembly of genomes from pooled samples of genetically heterogenous samples of conspecifics remains challenging. In this study, we show that high-quality genome assemblies can be produced from samples of multiple wild-caught individuals. We sequenced DNA extracted from a pooled sample of conspecific herbivorous insects (Hemiptera: Miridae: Tupiocoris notatus) acquired from a greenhouse infestation in Tucson, Arizona (in the range of 30-100 individuals; 0.5 mL tissue by volume) using PacBio highly accurate long reads (HiFi). The initial assembly contained multiple haplotigs (>85% BUSCOs duplicated), but duplicate contigs could be easily purged to reveal a highly complete assembly (95.6% BUSCO, 4.4% duplicated) that is highly contiguous by short-read assembly standards (N 50 = 675 kb; Largest contig = 4.3 Mb). We then used our assembly as the basis for a genome-guided differential expression study of host plant-specific transcriptional responses. We found thousands of genes (N = 4982) to be differentially expressed between our new data from individuals feeding on Datura wrightii (Solanaceae) and existing RNA-seq data from Nicotiana attenuata (Solanaceae)-fed individuals. We identified many of these genes as previously documented detoxification genes such as glutathione-S-transferases, cytochrome P450s, and UDP-glucosyltransferases. Together our results show that long-read sequencing of pooled samples can provide a cost-effective genome assembly option for small insects and can provide insights into the genetic mechanisms underlying interactions between plants and herbivorous pests.

The Complete and Gapless Genome Sequence of the Sugarbeet Pathogen Cercospora beticola

Cercospora beticola, causal agent of Cercospora leaf spot of sugarbeet ( Beta vulgaris subsp. vulgaris), is the most economically damaging foliar pathogen inhibiting sugarbeet production. Despite the global importance of C. beticola, significant knowledge gaps exist pertaining to pathogen virulence and adaptation to management practices that include the use of resistant host varieties and the application of different fungicide chemistries. The lack of a high-quality reference genome for C. beticola has hindered research progression addressing these critical knowledge gaps. Here, we report the complete and gapless genome assembly and annotation of the reference C. beticola isolate Cb09-40. We identified misassembled regions in the previous reference genome and annotated previously missing protein-coding genes. This work establishes a high-quality reference genome that can serve as the foundation for future comparative genomic, population genetic, and evolutionary studies into the virulence and evolution of this important plant pathogen. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

EcoGenoRisk: Developing a computational ecological risk assessment tool for synthetic biology

The expanding field of synthetic biology (synbio) supports new opportunities in the design of targeted bioproducts or modified microorganisms. However, this rapid development of synbio products raises concerns surrounding the potential risks of modified microorganisms contaminating unintended environments. These potential invasion risks require new bioinformatic tools to inform the design phase. EcoGenoRisk is a newly constructed computational risk assessment tool for invasiveness that aims to predict where synbio microorganisms may establish a population by screening for habitats of genetically similar microorganisms. The first module of the tool identifies genetically similar microorganisms and potential ecological relationships such as competition, mutualism, and inhibition. In total, 520 archaeal and 32,828 bacterial complete assembly genomes were analyzed to test the specificity and accuracy of the tool as well as to characterize the enzymatic profiles of different taxonomic lineages. Additionally, ecological relationships were analyzed to determine which would result in the greatest potential overlap between shared functional profiles. Notably, competition displayed the significantly highest overlap of shared functions between compared genomes. Overall, EcoGenoRisk is a flexible software pipeline that assists environmental risk assessors to query large databases of known microorganisms and prioritize follow-up bench scale studies.

Research on intelligent auxiliary assembly technology based on deep learning

Background Auxiliary assembly refers to guiding and prompting the assembly process to help operators complete complex assembly operations. Due to the complex structure of products, the similar shape of parts and human factors, the misassembly and missing assembly of parts still occur in the process of product assembly, so it is of great significance to detect the assembly correctness of complex products. Methods Aiming at the problem that manual inspection is inefficient and depends heavily on the level of inspectors in the process of complex product assembly inspection, this paper proposes an assembly correctness detection method based on deep learning. Through the three steps of view transformation, semantic segmentation and template matching, the automatic judgment of assembly errors such as wrong assembly, missing assembly and redundancy is realized, and the method is verified by the computer motherboard. Results Taking the computer motherboard as the verification object to test the correctness of assembly, the experimental re sults show that the perspective adjustment of the image after homography transformation is very obvious. The evaluation index of the semantic segmentation network detection object is calculated, and each accuracy meets the requirements of assembly correctness detection. A visualization module is also used to visually display the results of assembly correctness detection based on template matching. Conclusions The assembly correctness detection method can provide a guarantee for the manual assembly process and reduce the error rate of assembly. The machine vision detection technology can be used for automatic detection of assembly quality to improve the efficiency and automation level of detection.

The plasmidome associated with Gram-negative bloodstream infections: A large-scale observational study using complete plasmid assemblies

Plasmids carry genes conferring antimicrobial resistance and other clinically important traits, and contribute to the rapid dissemination of such genes. Previous studies using complete plasmid assemblies, which are essential for reliable inference, have been small and/or limited to plasmids carrying antimicrobial resistance genes (ARGs). In this study, we sequenced 1,880 complete plasmids from 738 isolates from bloodstream infections in Oxfordshire, UK. The bacteria had been originally isolated in 2009 (194 isolates) and 2018 (368 isolates), plus a stratified selection from intervening years (176 isolates). We demonstrate that plasmids are largely, but not entirely, constrained to a single host species, although there is substantial overlap between species of plasmid gene-repertoire. Most ARGs are carried by a relatively small number of plasmid groups with biological features that are predictable. Plasmids carrying ARGs (including those encoding carbapenemases) share a putative ‘backbone’ of core genes with those carrying no such genes. These findings suggest that future surveillance should, in addition to tracking plasmids currently associated with clinically important genes, focus on identifying and monitoring the dissemination of high-risk plasmid groups with the potential to rapidly acquire and disseminate these genes.

Complete genome assemblies of several Xylella fastidiosa subspecies multiplex strains reveals high phage content and novel plasmids

The Gram-negative bacterium Xylella fastidiosa (Xf) was originally found in the Americas, but has now been identified in more than 20 countries across America, Asia, and Europe. This plant pathogen is currently listed as a priority pest in Europe due to its socio-economic and ecological impacts. Within the three Xf subspecies fastidiosa, multiplex and pauca, subsp. multiplex displays a notably wider range of host plants than the other two subspecies. Comparative genomics may allow determination of how Xf subsp. multiplex adapts to new and diverse hosts and environments, so it is important that more genomes of this subspecies are defined. Twelve complete closed genomes sequences of Xf subsp. multiplex were obtained using a hybrid assembly approach combining Illumina and Oxford Nanopore technologies. The combined use of Canu and Unicycler assemblers enabled identification and closure of several plasmid sequences with high similarity to other plasmids described in strains of Xf subsp. fastidiosa and subsp. pauca. The analysis also revealed prophage sequences and contigs outside the chromosomes, annotated as phages. These new genomes, in conjunction with those existing in GenBank, will facilitate exploration of the evolutionary dynamics of Xf subsp. multiplex, its host adaptation mechanisms, and the potential emergence of novel strains of this important plant pathogen.

An intramolecular cross-talk in D29 mycobacteriophage endolysin governs the lytic cycle and phage-host population dynamics.

D29 mycobacteriophage encodes LysA endolysin, which mediates mycobacterial host cell lysis by targeting its peptidoglycan layer, thus projecting itself as a potential therapeutic. However, the regulatory mechanism of LysA during the phage lytic cycle remains ill defined. Here, we show that during D29 lytic cycle, structural and functional regulation of LysA not only orchestrates host cell lysis but also is critical for maintaining phage-host population dynamics by governing various phases of lytic cycle. We report that LysA exists in two conformations, of which only one is active, and the protein undergoes a host peptidoglycan-dependent conformational switch to become active for carrying out endogenous host cell lysis. D29 maintains a pool of inactive LysA, allowing complete assembly of phage progeny, thus helping avoid premature host lysis. In addition, we show that the switch reverses after lysis, thus preventing exogenous targeting of bystanders, which otherwise negatively affects phage propagation in the environment.

Extraction and selection of high-molecular-weight DNA for long-read sequencing from Chlamydomonas reinhardtii.

Recent advances in long-read sequencing technologies have enabled the complete assembly of eukaryotic genomes from telomere to telomere by allowing repeated regions to be fully sequenced and assembled, thus filling the gaps left by previous short-read sequencing methods. Furthermore, long-read sequencing can also help characterizing structural variants, with applications in the fields of genome evolution or cancer genomics. For many organisms, the main bottleneck to sequence long reads remains the lack of robust methods to obtain high-molecular-weight (HMW) DNA. For this purpose, we developed an optimized protocol to extract DNA suitable for long-read sequencing from the unicellular green alga Chlamydomonas reinhardtii, based on CTAB/phenol extraction followed by a size selection step for long DNA molecules. We provide validation results for the extraction protocol, as well as statistics obtained with Oxford Nanopore Technologies sequencing.

Structures of complete extracellular receptor assemblies mediated by IL-12 and IL-23.

Cell-surface receptor complexes mediated by pro-inflammatory interleukin (IL)-12 and IL-23, both validated therapeutic targets, are incompletely understood due to the lack of structural insights into their complete extracellular assemblies. Furthermore, there is a paucity of structural details describing the IL-12-receptor interaction interfaces, in contrast to IL-23-receptor complexes. Here we report structures of fully assembled mouse IL-12/human IL-23-receptor complexes comprising the complete extracellular segments of the cognate receptors determined by electron cryo-microscopy. The structures reveal key commonalities but also surprisingly diverse features. Most notably, whereas IL-12 and IL-23 both utilize a conspicuously presented aromatic residue on their α-subunit as a hotspot to interact with the N-terminal Ig domain of their high-affinity receptors, only IL-12 juxtaposes receptor domains proximal to the cell membrane. Collectively, our findings will help to complete our understanding of cytokine-mediated assemblies of tall cytokine receptors and will enable a cytokine-specific interrogation of IL-12/IL-23 signaling in physiology and disease.

PMAT: an efficient plant mitogenome assembly toolkit using low-coverage HiFi sequencing data.

Complete mitochondrial genomes (mitogenomes) of plants are valuable resources for nucleocytoplasmic interactions, plant evolution, and plant cytoplasmic male sterile line breeding. However, the complete assembly of plant mitogenomes is challenging due to frequent recombination events and horizontal gene transfers. Previous studies have adopted Illumina, PacBio, and Nanopore sequencing data to assemble plant mitogenomes, but the poor assembly completeness, low sequencing accuracy, and high cost limit the sampling capacity. Here, we present an efficient assembly toolkit (PMAT) for de novo assembly of plant mitogenomes using low-coverage HiFi sequencing data. PMAT has been applied to the de novo assembly of 13 broadly representative plant mitogenomes, outperforming existing organelle genome assemblers in terms of assembly accuracy and completeness. By evaluating the assembly of plant mitogenomes from different sequencing data, it was confirmed that PMAT only requires 1× HiFi sequencing data to obtain a complete plant mitogenome. The source code for PMAT is available at https://github.com/bichangwei/PMAT. The developed PMAT toolkit will indeed accelerate the understanding of evolutionary variation and breeding application of plant mitogenomes.

A haplotype-resolved gap-free genome assembly provides novel insight into monoterpenoid diversification in Mentha suaveolens 'Variegata'.

Mentha is a commonly used spice worldwide, which possesses medicinal properties and fragrance. These characteristics are conferred, at least partially, by essential oils such as menthol. In this study, a gap-free assembly with a genome size of 414.3Mb and 31,251 coding genes was obtained for Mentha suaveolens 'Variegata'. Based on its high heterozygosity (1.5%), two complete haplotypic assemblies were resolved, with genome sizes of 401.9 and 405.7Mb, respectively. The telomeres and centromeres of each haplotype were almost fully annotated. In addition, we detected a total of 41,135 structural variations. Enrichment analysis demonstrated that genes involved in terpenoid biosynthesis were affected by these structural variations. Analysis of volatile metabolites showed that M. suaveolens mainly produces piperitenone oxide rather than menthol. We identified three genes in the M. suaveolens genome which encode isopiperitenone reductase (ISPR), a key rate-limiting enzyme in menthol biosynthesis. However, the transcription levels of ISPR were low. Given that other terpenoid biosynthesis genes were expressed, M. suaveolens ISPRs may account for the accumulation of piperitenone oxide in this species. The findings of this study may provide a valuable resource for improving the detection rate and accuracy of genetic variants, thereby enhancing our understanding of their impact on gene function and expression. Moreover, our haplotype-resolved gap-free genome assembly offers novel insights into molecular marker-assisted breeding of Mentha.

An effective strategy for assembling the sex-limited chromosome.

Most currently available reference genomes lack the sequence map of sex-limited (such as Y and W) chromosomes, which results in incomplete assemblies that hinder further research on sex chromosomes. Recent advancements in long-read sequencing and population sequencing have provided the opportunity to assemble sex-limited chromosomes without the traditional complicated experimental efforts. We introduce the first computational method, Sorting long Reads of Y or other sex-limited chromosome (SRY), which achieves improved assembly results compared to flow sorting. Specifically, SRY outperforms in the heterochromatic region and demonstrates comparable performance in other regions. Furthermore, SRY enhances the capabilities of the hybrid assembly software, resulting in improved continuity and accuracy. Our method enables true complete genome assembly and facilitates downstream research of sex-limited chromosomes.

Genome Resource of Raspberry Root Rot Pathogen Phytophthora gonapodyides.

Phytophthora gonapodyides is a newly reported oomycetes pathogen associated with root rot of red raspberry. We generated high-quality whole genome resource for P. gonapodyides, which was pathogenic on red raspberry. The genome size was 88,717,598 bp with a BUSCO completeness score of 93.9%. This genome resource provides insight on pathogen biology of Phytophthora spp. causing root rot of raspberry. To our best knowledge, this is the first complete genome assembly of plant pathogenic P. gonapodyides.

A Generative Shape Compositional Framework to Synthesize Populations of Virtual Chimeras.

Generating virtual organ populations that capture sufficient variability while remaining plausible is essential to conduct in silico trials (ISTs) of medical devices. However, not all anatomical shapes of interest are always available for each individual in a population. The imaging examinations and modalities used can vary between subjects depending on their individualized clinical pathways. Different imaging modalities may have various fields of view and are sensitive to signals from other tissues/organs, or both. Hence, missing/partially overlapping anatomical information is often available across individuals. We introduce a generative shape model for multipart anatomical structures, learnable from sets of unpaired datasets, i.e., where each substructure in the shape assembly comes from datasets with missing or partially overlapping substructures from disjoint subjects of the same population. The proposed generative model can synthesize complete multipart shape assemblies coined virtual chimeras (VCs). We applied this framework to build VCs from databases of whole-heart shape assemblies that each contribute samples for heart substructures. Specifically, we propose a graph neural network-based generative shape compositional framework, which comprises two components, a part-aware generative shape model that captures the variability in shape observed for each structure of interest in the training population and a spatial composition network that assembles/composes the structures synthesized by the former into multipart shape assemblies (i.e., VCs). We also propose a novel self-supervised learning scheme that enables the spatial composition network to be trained with partially overlapping data and weak labels. We trained and validated our approach using shapes of cardiac structures derived from cardiac magnetic resonance (MR) images in the UK Biobank (UKBB). When trained with complete and partially overlapping data, our approach significantly outperforms a principal component analysis (PCA)-based shape model (trained with complete data) in terms of generalizability and specificity. This demonstrates the superiority of the proposed method, as the synthesized cardiac virtual populations are more plausible and capture a greater degree of shape variability than those generated by the PCA-based shape model.

Reference Genome Resource for the Citrus Pathogen Phytophthora citrophthora.

Phytophthora citrophthora is an oomycete pathogen that infects citrus. Its occurrence in citrus-growing regions worldwide is considered a major contributor to crop losses. This study presents a high-quality genome resource for P. citrophthora, which was generated using PacBio HiFi long-read high-throughput sequencing technology. We successfully assembled a 48.5 Mb genome containing 16,409 protein-coding genes from high-quality reads. This marks the first complete genome assembly of P. citrophthora, providing a valuable resource to enhance the understanding of pathogenic behaviour and fungicide sensitivity of this destructive citrus pathogen.

Small-Angle X-Ray Scattering for Macromolecular Complexes.

Small angle X-ray scattering (SAXS) is a versatile technique that can provide unique insights in the solution structure of macromolecules and their complexes, covering the size range from small peptides to complete viral assemblies. Technological and conceptual advances in the last two decades have tremendously improved the accessibility of the technique and transformed it into an indispensable tool for structural biology. In this chapter we introduce and discuss several approaches to collecting SAXS data on macromolecular complexes, including several approaches to online chromatography. We include practical advice on experimental design and point out common pitfalls of the technique.

Comparisons of genome assembly tools for characterization of Mycobacterium tuberculosis genomes using hybrid sequencing technologies.

Next-generation sequencing of Mycobacterium tuberculosis, the infectious agent causing tuberculosis, is improving the understanding of genomic diversity of circulating lineages and strain-types, and informing knowledge of drug resistance mutations. An increasingly popular approach to characterizing M. tuberculosis genomes (size: 4.4 Mbp) and variants (e.g., single nucleotide polymorphisms (SNPs)) involves the de novo assembly of sequence data. We compared the performance of genome assembly tools (Unicycler, RagOut, and RagTag) on sequence data from nine drug resistant M. tuberculosis isolates (multi-drug (MDR) n = 1; pre-extensively-drug (pre-XDR) n = 8) generated using Illumina HiSeq, Oxford Nanopore Technology (ONT) PromethION, and PacBio platforms. Our investigation found that Unicycler-based assemblies had significantly higher genome completeness (~98.7%; p values = 0.01) compared to other assembler tools (RagOut = 98.6%, and RagTag = 98.6%). The genome assembly sizes (bp) across isolates and sequencers based on RagOut was significantly longer (p values < 0.001) (4,418,574 ± 8,824 bp) than Unicycler and RagTag assemblies (Unicycler = 4,377,642 ± 55,257 bp, and RagTag = 4,380,711 ± 51,164 bp). RagOut-based assemblies had the fewest contigs (~32) and the longest genome size (4,418,574 bp; vs. H37Rv reference size 4,411,532 bp) and therefore were chosen for downstream analysis. Pan-genome analysis of Illumina and PacBio hybrid assemblies revealed the greatest number of detected genes (4,639 genes; H37Rv reference contains 3,976 genes), while Illumina and ONT hybrid assemblies produced the highest number of SNPs. The number of genes from hybrid assemblies with ONT and PacBio long-reads (mean: 4,620 genes) was greater than short-read assembly alone (4,478 genes). All nine RagOut hybrid genome assemblies detected known mutations in genes associated with MDR-TB and pre-XDR-TB. Unicycler software performed the best in terms of achieving contiguous genomes, whereas RagOut improved the quality of Unicycler's genome assemblies by providing a longer genome size. Overall, our approach has demonstrated that short-read and long-read hybrid assembly can provide a more complete genome assembly than short-read assembly alone by detecting pan-genomes and more genes, including IS6110, and SNPs.