Final Assemblies Research Articles

Mass customization using hybrid manufacturing and smart assembly: An optimal configuration and platform design approach

Hybrid Manufacturing (HM) and smart assembly stand as pivotal pillars in advanced smart manufacturing systems, offering manufacturers highly efficient and adaptable solutions for manufacturing. This paper delves into the configuration of a production line that integrates HM and assembly stages, each comprising multiple cells, with each cell housing one or more parallel stations. The objective is to manufacture a family of final assemblies, leveraging the platform concept to defer mass customization to later stages and thereby minimize processing costs. A mathematical programming model is proposed to identify the optimal configuration for such production lines, considering constraints such as an allowable capital cost and machine availabilities. In addition, the precedence, inclusion, and seclusion restrictions imposed on the part family are considered. The proposed mathematical programming model aims to delineate which HM features are processed in the part platform cell versus those processed in the mass customization (part variants) cells. Simultaneously, the model determines the components (variants from the HM stage) of final assemblies processed in the assembly platform cell, as well as components assembled or disassembled in the final assembly cells. Furthermore, the model seeks to determine the required number of stations in each cell to meet periodic demand. The overall objective of the model is to minimize the capital and the processing cost. A detailed case study illustrates the effectiveness of the proposed configuration approach and mathematical model. The proposed model is solvable in a few seconds by using commercial solvers.

Cascade–Assembly–Confined Multilevel Supramolecular Assemblies for Multicolor Luminescence Information Storage and Cells Imaging Based on Slipped Pseudo[5]rotaxane

Multilevel green fluorescent supramolecular assemblies that derived from macrocyclic confinement–activated yellow fluorescent pseudo[5]rotaxane and subsequently assembled with anionic cyclodextrin derivative sulfobutylether–β–cyclodextrin (SBE–β–CD) were reported. Possessing four cationic coumarin arms, the coumarin–modified dibenzo–24–crown–8 guest (G) could be encapsulated by cucurbit[7]uril (CB[7]) at a 4:1 host–guest stoichiometry to form pseudo[5]rotaxane (G@CB[7]4) in the manner of dynamic slipping, which not only extended the fluorescence intensity of G by 53 times, but also increased the fluorescence lifetime by 5.8–fold from 0.65 to 3.76 ns. Subsequently, SBE–β–CD was co–assembled with G@CB[7]4 to control the topological morphology from small nanoparticles to larger ones, further enhancing fluorescence by 9.9 times along with a blue shift from 530 nm to 515 nm through cascade confinement. Compared with the direct assembly of G and SBE–β–CD through electrostatic interactions, only slight fluorescence increase was shown. After doping the NIR dye sulforhodamine 101 (SR101) acceptor, the G@CB[7]4@SBE–β–CD/SR101 exhibited high energy transfer efficiency to 79%, accompanied by the color changed from green to red under 365 nm irradiation. Moreover, the multicolor tunable emission could be achieved by adding dibenzylammonium chloride to the solution of G@CB[7]4, G@CB[7]4@SBE–β–CD and G@CB[7]4@SBE–β–CD/SR101. Ultimately, the final assemblies were used in multicolor luminescence information storage and cells imaging.

Complex Pathways Drive Pluripotent Fmoc-Leucine Self-Assemblies.

Nature uses complex self-assembly pathways to access distinct functional non-equilibrium self-assemblies. This remarkable ability to steer same set of biomolecules into different self-assembly states is done by avoiding thermodynamic pit. In synthetic systems, on demand control over 'Pathway Complexity' to access self-assemblies different from equilibrium structures remains challenging. Here we show versatile non-equilibrium assemblies of the same monomer via alternate assembly pathways. The assemblies nucleate using non-classical or classical nucleation routes into distinct metastable (transient hydrogels), kinetic (stable hydrogels) and thermodynamic structures [(poly)-crystals and 2D sheets]. Initial chemical and thermal inputs force the monomers to follow different assembly pathways and form soft-materials with distinct molecular arrangements than at equilibrium. In many cases, equilibrium structures act as thermodynamic sink which consume monomers from metastable structures giving transiently formed materials. This dynamics can be tuned chemically or thermally to slow down the dissolution of transient hydrogel, or skip the intermediate hydrogel altogether to reach final equilibrium assemblies. If required this metastable state can be kinetically trapped to give strong hydrogel stable over days. This method to control different self-assembly states can find potential use in similar biomimetic systems to access new materials for various applications.

Complex Pathways Drive Pluripotent Fmoc‐Leucine Self‐Assemblies

AbstractNature uses complex self‐assembly pathways to access distinct functional non‐equilibrium self‐assemblies. This remarkable ability to steer same set of biomolecules into different self‐assembly states is done by avoiding thermodynamic pit. In synthetic systems, on demand control over ‘Pathway Complexity’ to access self‐assemblies different from equilibrium structures remains challenging. Here we show versatile non‐equilibrium assemblies of the same monomer via alternate assembly pathways. The assemblies nucleate using non‐classical or classical nucleation routes into distinct metastable (transient hydrogels), kinetic (stable hydrogels) and thermodynamic structures [(poly)‐crystals and 2D sheets]. Initial chemical and thermal inputs force the monomers to follow different assembly pathways and form soft‐materials with distinct molecular arrangements than at equilibrium. In many cases, equilibrium structures act as thermodynamic sink which consume monomers from metastable structures giving transiently formed materials. This dynamics can be tuned chemically or thermally to slow down the dissolution of transient hydrogel, or skip the intermediate hydrogel altogether to reach final equilibrium assemblies. If required this metastable state can be kinetically trapped to give strong hydrogel stable over days. This method to control different self‐assembly states can find potential use in similar biomimetic systems to access new materials for various applications.

Whole-genome sequencing of Ganoderma boninense, the causal agent of basal stem rot disease in oil palm, via combined short- and long-read sequencing

The hemibiotrophic Basidiomycete pathogen Ganoderma boninense (Gb) is the dominant causal agent of oil palm basal stem rot disease. Here, we report a complete chromosomal genome map of Gb using a combination of short-read Illumina and long-read Pacific Biosciences (PacBio) sequencing platforms combined with chromatin conformation capture data from the Chicago and Hi-C platforms. The genome was 55.87 Mb in length and assembled to a high contiguity (N50: 304.34 kb) of 12 chromosomes built from 112 scaffolds, with a total of only 4.34 Mb (~ 7.77%) remaining unplaced. The final assemblies were evaluated for completeness of the genome by using Benchmarking Universal Single Copy Orthologs (BUSCO) v4.1.4, and based on 4464 total BUSCO polyporales group searches, the assemblies yielded 4264 (95.52%) of the conserved orthologs as complete and only a few fragmented BUSCO of 42 (0.94%) as well as a missing BUSCO of 158 (3.53%). Genome annotation predicted a total of 21,074 coding genes, with a GC content ratio of 59.2%. The genome features were analyzed with different databases, which revealed 2471 Gene Ontology/GO (11.72%), 5418 KEGG (Kyoto Encyclopedia of Genes and Genomes) Orthologous/KO (25.71%), 13,913 Cluster of Orthologous Groups of proteins/COG (66.02%), 60 ABC transporter (0.28%), 1049 Carbohydrate-Active Enzymes/CAZy (4.98%), 4005 pathogen–host interactions/PHI (19%), and 515 fungal transcription factor/FTFD (2.44%) genes. The results obtained in this study provide deep insight for further studies in the future.

Comparative Genomic Analysis of Colletotrichum lini Strains with Different Virulence on Flax.

Colletotrichum lini is a flax fungal pathogen. The genus comprises differently virulent strains, leading to significant yield losses. However, there were no attempts to investigate the molecular mechanisms of C. lini pathogenicity from high-quality genome assemblies until this study. In this work, we sequenced the genomes of three C. lini strains of high (#390-1), medium (#757), and low (#771) virulence. We obtained more than 100× genome coverage with Oxford Nanopore Technologies reads (N50 = 12.1, 6.1, 5.0 kb) and more than 50× genome coverage with Illumina data (150 + 150 bp). Several assembly strategies were tested. The final assemblies were obtained using the Canu-Racon ×2-Medaka-Polca scheme. The assembled genomes had a size of 54.0-55.3 Mb, 26-32 contigs, N50 values > 5 Mb, and BUSCO completeness > 96%. A comparative genomic analysis showed high similarity among mitochondrial and nuclear genomes. However, a rearrangement event and the loss of a 0.7 Mb contig were revealed. After genome annotation with Funannotate, secreting proteins were selected using SignalP, and candidate effectors were predicted among them using EffectorP. The analysis of the InterPro annotations of predicted effectors revealed unique protein categories in each strain. The assembled genomes and the conducted comparative analysis extend the knowledge of the genetic diversity of C. lini and form the basis for establishing the molecular mechanisms of its pathogenicity.

A Whole-Genome Sequencing-Based Approach for the Characterization of Klebsiella pneumoniae Co-Producing KPC and OXA-48-like Carbapenemases Circulating in Sardinia, Italy.

Whole-genome sequencing (WGS) provides important information for the characterization, surveillance, and monitoring of antimicrobial resistance (AMR) determinants, particularly in cases of multi- and extensively drug-resistant microorganisms. We reported the results of a WGS analysis carried out on carbapenemases-producing Klebsiella pneumoniae, which causes hospital-acquired infections (HAIs) and is characterized by a marked resistance profile. Clinical, phenotypic, and genotypic data were collected for the AMR surveillance screening program of the University Hospital of Sassari (Italy) during 2020-2021. Genomic DNA was sequenced using the Illumina Nova Seq 6000 platform. Final assemblies were manually curated and carefully verified for the detection of antimicrobial resistance genes, porin mutations, and virulence factors. A phylogenetic analysis was performed using the maximum likelihood method. All 17 strains analyzed belonged to ST512, and most of them carried the blaKPC-31 variant blaOXA-48-like, an OmpK35 truncation, and an OmpK36 mutation. Phenotypic analysis showed a marked resistance profile to all antibiotic classes, including β-lactams, carbapenems, aminoglycosides, fluoroquinolone, sulphonamides, and novel β-lactam/β-lactamase inhibitors (BL/BLI). WGS characterization revealed the presence of several antibiotic resistance determinants and porin mutations in highly resistant K. pneumoniae strains responsible for HAIs. The detection of blaKPC-31 in our hospital wards highlights the importance of genomic surveillance in hospital settings to monitor the emergence of new clones and the need to improve control and preventive strategies to efficiently contrast AMR.

Supramolecular assembly guided by photolytic redox cycling

AbstractIn living systems, the formation of structures relies on balancing kinetic and thermodynamic influences powered by reversible covalent bond chemistry. Although synthetic efforts have replicated these processes to some extent, elucidating their combination is necessary to identify mechanisms that confer nature’s structural precision and flexibility within a complex environment. Here we design a photolytic reaction cascade where competing redox pathways control the transience, interconversion and production rates of thiol/disulfide supramolecular monomers in situ. In contrast to direct assembly by dissolution, cascade generation of the same monomers formed hierarchical assemblies with different structural order. Redox-induced cycling between thiol–disulfide formation led to the emergence of new secondary structures and chirality within the final assemblies. These multiple structural states found within the same molecular system demonstrate the concept of assembly plasticity engaged frequently in biology. We demonstrate the importance of reaction complexity in controlling supramolecular propagation and in expanding the library of nanoarchitectures that can be created.

DNA Materials Assembled from One DNA Strand.

Due to the specific base-pairing recognition, clear nanostructure, programmable sequence and responsiveness of the DNA molecule, DNA materials have attracted extensive attention and been widely used in controlled release, drug delivery and tissue engineering. Generally, the strategies for preparing DNA materials are based on the assembly of multiple DNA strands. The construction of DNA materials using only one DNA strand can not only save time and cost, but also avoid defects in final assemblies generated by the inaccuracy of DNA ratios, which potentially promote the large-scale production and practical application of DNA materials. In order to use one DNA strand to form assemblies, the sequences have to be palindromes with lengths that need to be controlled carefully. In this review, we introduced the development of DNA assembly and mainly summarized current reported materials formed by one DNA strand. We also discussed the principle for the construction of DNA materials using one DNA strand.

Draft Genome Assembly of Stutzerimonas sp. Strain S1 and Achromobacter spanius Strain S4, Two Syringol-Metabolizing Bacteria Isolated from Compost Soil.

Two bacterial strains able to use syringol as a sole carbon source were isolated from compost. The isolates, named S1 and S4, were sequenced using the Illumina platform. The final assemblies contained 4.2 Mbp, 63% GC, and 3,912 genes for S1 and 6.2 Mbp, 64% GC, and 5,503 genes for S4.

Influence of Centrifugation and Shaking on the Self-Assembly of Lysozyme Fibrils.

Protein self-assembly into fibrils and oligomers plays a key role in the etiology of degenerative diseases. Several pathways for this self-assembly process have been described and shown to result in different types and ratios of final assemblies, therewith defining the effective physiological response. Known factors that influence assembly pathways are chemical conditions and the presence or lack of agitation. However, in natural and industrial systems, proteins are exposed to a sequence of different and often complex mass transfers. In this paper, we compare the effect of two fundamentally different mass transfer processes on the fibrilization process. Aggregation-prone solutions of hen egg white lysozyme were subjected to predominantly non-advective mass transfer by employing centrifugation and to advective mass transport represented by orbital shaking. In both cases, fibrilization was triggered, while in quiescent only oligomers were formed. The fibrils obtained by shaking compared to fibrils obtained through centrifugation were shorter, thicker, and more rigid. They had rod-like protofibrils as building blocks and a significantly higher β-sheet content was observed. In contrast, fibrils from centrifugation were more flexible and braided. They consisted of intertwined filaments and had low β-sheet content at the expense of random coil. To the best of our knowledge, this is the first evidence of a fibrilization pathway selectivity, with the fibrilization route determined by the mass transfer and mixing configuration (shaking versus centrifugation). This selectivity can be potentially employed for directed protein fibrilization.

Reconstruction of Acinetobacter johnsonii ICE_NC genome using hybrid de novo genome assemblies and identification of the 12α-hydroxysteroid dehydrogenase gene.

AimsThe role of a Acinetobacter johnsonii strain, isolated from a soil sample, in the biotransformation of bile acids (BAs) was already described but the enzymes responsible for these transformations were only partially purified and molecularly characterized.Methods and ResultsThis study describes the use of hybrid de novo assemblies, that combine long‐read Oxford Nanopore and short‐read Illumina sequencing strategies, to reconstruct the entire genome of A. johnsonii ICE_NC strain and to identify the coding region for a 12α‐hydroxysteroid dehydrogenase (12α‐HSDH), involved in BAs metabolism. The de novo assembly of the A. johnsonii ICE_NC genome was generated using Canu and Unicycler, both strategies yielded a circular chromosome of about 3.6 Mb and one 117 kb long plasmid. Gene annotation was performed on the final assemblies and the gene for 12α‐HSDH was detected on the plasmid.ConclusionsOur findings illustrate the added value of long read sequencing in addressing the challenges of whole genome characterization and plasmid reconstruction in bacteria. These approaches also allowed the identification of the A. johnsonii ICE_NC gene for the 12α‐HSDH enzyme, whose activity was confirmed at the biochemical level.Significance and impact or the studyAt present, this is the first report on the characterization of a 12α‐HSDH gene in an A. johnsonii strain able to biotransform cholic acid into ursodeoxycholic acid, a promising therapeutic agent for several diseases.

Recovering individual haplotypes and a contiguous genome assembly from pooled long-read sequencing of the diamondback moth (Lepidoptera: Plutellidae).

The assembly of divergent haplotypes using noisy long-read data presents a challenge to the reconstruction of haploid genome assemblies, due to overlapping distributions of technical sequencing error, intralocus genetic variation, and interlocus similarity within these data. Here, we present a comparative analysis of assembly algorithms representing overlap-layout-consensus, repeat graph, and de Bruijn graph methods. We examine how postprocessing strategies attempting to reduce redundant heterozygosity interact with the choice of initial assembly algorithm and ultimately produce a series of chromosome-level assemblies for an agricultural pest, the diamondback moth, Plutella xylostella (L.). We compare evaluation methods and show that BUSCO analyses may overestimate haplotig removal processing in long-read draft genomes, in comparison to a k-mer method. We discuss the trade-offs inherent in assembly algorithm and curation choices and suggest that “best practice” is research question dependent. We demonstrate a link between allelic divergence and allele-derived contig redundancy in final genome assemblies and document the patterns of coding and noncoding diversity between redundant sequences. We also document a link between an excess of nonsynonymous polymorphism and haplotigs that are unresolved by assembly or postassembly algorithms. Finally, we discuss how this phenomenon may have relevance for the usage of noisy long-read genome assemblies in comparative genomics.

The gill transcriptome of threatened European freshwater mussels

Genomic tools applied to non-model organisms are critical to design successful conservation strategies of particularly threatened groups. Freshwater mussels of the Unionida order are among the most vulnerable taxa and yet almost no genetic resources are available. Here, we present the gill transcriptomes of five European freshwater mussels with high conservation concern: Margaritifera margaritifera, Unio crassus, Unio pictorum, Unio mancus and Unio delphinus. The final assemblies, with N50 values ranging from 1069–1895 bp and total BUSCO scores above 90% (Eukaryote and Metazoan databases), were structurally and functionally annotated, and made available. The transcriptomes here produced represent a valuable resource for future studies on these species’ biology and ultimately guide their conservation.

Pan-genome analysis of three main Chinese chestnut varieties.

Chinese chestnut (Castanea mollissima Blume) is one of the earliest domesticated plants of high nutritional and ecological value, yet mechanisms of C. mollissima underlying its growth and development are poorly understood. Although individual chestnut species differ greatly, the molecular basis of the formation of their characteristic traits remains unknown. Though the draft genomes of chestnut have been previously released, the pan-genome of different variety needs to be studied. We report the genome sequence of three cultivated varieties of chestnut herein, namely Hei-Shan-Zhai-7 (H7, drought-resistant variety), Yan-Hong (YH, easy-pruning variety), and Yan-Shan-Zao-Sheng (ZS, early-maturing variety), to expedite convenience and efficiency in its genetics-based breeding. We obtained three chromosome-level chestnut genome assemblies through a combination of Oxford Nanopore technology, Illumina HiSeq X, and Hi-C mapping. The final genome assemblies are 671.99 Mb (YH), 790.99 Mb (ZS), and 678.90 Mb (H7), across 12 chromosomes, with scaffold N50 sizes of 50.50 Mb (YH), 65.05 Mb (ZS), and 52.16 Mb (H7). Through the identification of homologous genes and the cluster analysis of gene families, we found that H7, YH and ZS had 159, 131, and 91 unique gene families, respectively, and there were 13,248 single-copy direct homologous genes in the three chestnut varieties. For the convenience of research, the chestnut genome database1 was constructed. Based on the results of gene family identification, the presence/absence variations (PAVs) information of the three sample genes was calculated, and a total of 2,364, 2,232, and 1,475 unique genes were identified in H7, YH and ZS, respectively. Our results suggest that the GBSS II-b gene family underwent expansion in chestnut (relative to nearest source species). Overall, we developed high-quality and well-annotated genome sequences of three C. mollissima varieties, which will facilitate clarifying the molecular mechanisms underlying important traits, and shortening the breeding process.

Targeted de novo phasing and long-range assembly by template mutagenesis.

Short-read sequencers provide highly accurate reads at very low cost. Unfortunately, short reads are often inadequate for important applications such as assembly in complex regions or phasing across distant heterozygous sites. In this study, we describe novel bench protocols and algorithms to obtain haplotype-phased sequence assemblies with ultra-low error for regions 10 kb and longer using short reads only. We accomplish this by imprinting each template strand from a target region with a dense and unique mutation pattern. The mutation process randomly and independently converts ∼50% of cytosines to uracils. Sequencing libraries are made from both mutated and unmutated templates. Using de Bruijn graphs and paired-end read information, we assemble each mutated template and use the unmutated library to correct the mutated bases. Templates are partitioned into two or more haplotypes, and the final haplotypes are assembled and corrected for residual template mutations and PCR errors. With sufficient template coverage, the final assemblies have per-base error rates below 10–9. We demonstrate this method on a four-member nuclear family, correctly assembling and phasing three genomic intervals, including the highly polymorphic HLA-B gene.

Genomes of single- and double-petal jasmines (Jasminum sambac) provide insights into their divergence time and structural variations.

Genomes of single- and double-petal jasmines (Jasminum sambac) provide insights into their divergence time and structural variations.

Classification of emerging patterns in self-assembled two-dimensional magnetic lattices.

Self-assembled granular materials can be utilized in many applications such as shock absorption and energy harvesting. Such materials are inherently discrete with an easy path to tunability through external applied forces such as stress or by adding more elements to the system. However, the self-assembly process is statistical in nature with no guarantee for repeatability, stability, or order of emergent final assemblies. Here we study both numerically and experimentally the two-dimensional self-assembly of free-floating disks with repulsive magnetic potentials confined to a boundary with embedded permanent magnets. Six different types of disks and seven boundary shapes are considered. An agent-based model is developed to predict the self-assembled patterns for any given disk type, boundary, and number of disks. The validity of the model is experimentally verified. While the model converges to a physical solution, these solutions are not always unique and depend on the initial position of the disks. The emerging patterns are classified into monostable patterns (i.e., stable patterns that emerge regardless of the initial conditions) and multistable patterns. We also characterize the emergent order and crystallinity of the emerging patterns. The developed model along with the self-assembly nature of the system can be key in creating re-programmable materials with exceptional nonlinear properties.

Nematode Genome Announcement: Draft Genome of Meloidogyne chitwoodi, an Economically Important Pest of Potato in the Pacific Northwest

Meloidogyne chitwoodi is one of the most devastating pests of potato in the U.S. Pacific Northwest (PNW). Nematode-infected tubers develop external as well as internal defects, making the potato tubers unmarketable, and resulting in economic losses. Draft genome assemblies of three M. chitwoodi genotypes—race 1, race 2 and race 1 pathotype Roza—were generated using Illumina and PacBio Sequel RS II sequencing. The final assemblies consist of 30, 39, and 38 polished contigs for race 1, race 2 and race 1 pathotype Roza, respectively, with average N50 of 2.37 Mb and average assembled genome size of approximately 47.41 Mb. On average, 10,508 genes were annotated for each genome. Benchmarking universal single-copy ortholog (BUSCO) analysis indicated that 69.80% of the BUSCOs were complete whereas 68.80, 0.93, and 12.67% were single copy, duplicated, and fragmented, respectively. These highly contiguous genomes will enrich resources to study potato–nematode interactions and enhance breeding efforts to develop nematode-resistant potato varieties for the PNW. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Metatranscriptomic Analysis of Bacterial Communities on Laundered Textiles: A Pilot Case Study.

Microbially contaminated washing machines and mild laundering conditions facilitate the survival and growth of microorganisms on laundry, promoting undesired side effects such as malodor formation. Clearly, a deeper understanding of the functionality and hygienic relevance of the laundry microbiota necessitates the analysis of the microbial gene expression on textiles after washing, which—to the best of our knowledge—has not been performed before. In this pilot case study, we used single-end RNA sequencing to generate de novo transcriptomes of the bacterial communities remaining on polyester and cotton fabrics washed in a domestic washing machine in mild conditions and subsequently incubated under moist conditions for 72 h. Two common de novo transcriptome assemblers were used. The final assemblies included 22,321 Trinity isoforms and 12,600 Spades isoforms. A large part of these isoforms could be assigned to the SwissProt database, and was further categorized into “molecular function”, “biological process” and “cellular component” using Gene Ontology (GO) terms. In addition, differential gene expression was used to show the difference in the pairwise comparison of the two tissue types. When comparing the assemblies generated with the two assemblers, the annotation results were relatively similar. However, there were clear differences between the de novo assemblies regarding differential gene expression.