Initial Assembly Research Articles

Generation of elastic geodesic gridshells with anisotropic cross sections

Geodesic gridshells are shell structures made of continuous elements following geodesic lines. Their properties ease the use of beams with anisotropic cross-sections by avoiding bending about their strong axis. However, such bending may arise when flattening arbitrary geodesic grids, which forbids their initial assembly on the ground. This study provides a process to design elastic geodesic gridshells, that is, gridshells that minimise bending moments in both formed and near-flat configurations. The generation process first brings a target geodesic network onto a plane by maintaining arc lengths. The flat mesh is then relaxed to minimise its main curvatures and hence bending moments in its members. The result is an elastic geodesic gridshell that can be assembled flat on the ground and then lifted up into its target surface. The method is applied to the design of six geodesic gridshells made of reclaimed skis.

LongStitch: high-quality genome assembly correction and scaffolding using long reads

BackgroundGenerating high-quality de novo genome assemblies is foundational to the genomics study of model and non-model organisms. In recent years, long-read sequencing has greatly benefited genome assembly and scaffolding, a process by which assembled sequences are ordered and oriented through the use of long-range information. Long reads are better able to span repetitive genomic regions compared to short reads, and thus have tremendous utility for resolving problematic regions and helping generate more complete draft assemblies. Here, we present LongStitch, a scalable pipeline that corrects and scaffolds draft genome assemblies exclusively using long reads.ResultsLongStitch incorporates multiple tools developed by our group and runs in up to three stages, which includes initial assembly correction (Tigmint-long), followed by two incremental scaffolding stages (ntLink and ARKS-long). Tigmint-long and ARKS-long are misassembly correction and scaffolding utilities, respectively, previously developed for linked reads, that we adapted for long reads. Here, we describe the LongStitch pipeline and introduce our new long-read scaffolder, ntLink, which utilizes lightweight minimizer mappings to join contigs. LongStitch was tested on short and long-read assemblies of Caenorhabditis elegans, Oryza sativa, and three different human individuals using corresponding nanopore long-read data, and improves the contiguity of each assembly from 1.2-fold up to 304.6-fold (as measured by NGA50 length). Furthermore, LongStitch generates more contiguous and correct assemblies compared to state-of-the-art long-read scaffolder LRScaf in most tests, and consistently improves upon human assemblies in under five hours using less than 23 GB of RAM.ConclusionsDue to its effectiveness and efficiency in improving draft assemblies using long reads, we expect LongStitch to benefit a wide variety of de novo genome assembly projects. The LongStitch pipeline is freely available at https://github.com/bcgsc/longstitch.

Factors Controlling the Defect Formation in Cu-Sn Micro Joints in 2.5D and 3D Interconnects

The Cu-Sn reaction upon soldering has been thoroughly studied and the sporadic formation of Kirkendall voids within the intermetallic bonds between ball grid array (BGA) or surface mount technology (SMT) solder joints and Cu pads has been demonstrated as well-known concern. However, in the realm of ever developing device miniaturization, there is very limited information on the impact of space confinement on the dynamics of the Cu/solder joint interface. It is known that no voiding has been found in solder joints with high-purity Cu but this phenomenon presents itself, albeit to a different extent, when the joint is realized with electroplated Cu. Also, it is believed, that the voiding propensity in the Cu3Sn intermetallic compounds (IMC) is more likely to be higher in micro joints than in BGA/CSP scale (macro) joints for a given quality of electroplated Cu. Along with that it has been shown that the IMCs on Cu surfaces seem to grow much faster when the solder becomes very thin, and this is accompanied with a greatly enhanced risk of Kirkendall void nucleation.The emphasis of this work is on understanding the voiding behavior within the intermetallic bonds of Cu-Sn micro joints (5 – 30 µm) with the eventual goal of learning how it can be controlled or prevented. Our systematic studies have corroborated the association of voiding with the incorporation of small concentrations of impurities resulting from organic additives degradation during the Cu electroplating as already illustrated for macro joints. Our results also show lack of any voiding not only in macro- but also in micro joints when the interconnection is realized with high purity Cu, likely because no organic impurities are present in sufficient amount to prompt defect nucleation and growth. Our research further demonstrates that the IMC growth rate varies with factors and parameters such as solder volume to pad area ratio, solder composition, pad finish, initial assembly process parameters, subsequent heat treatments, and specifics of the further thermomechanical history, thus impacting differently the overall voiding level. Following this lead, the analysis of micro joints subjected to different synthetic and processing treatments clearly demonstrates the propensity for voiding to be substantially higher, for a given quality of electroplated Cu, in very small solder joints (5 – 30 µm), such as those found in 3D assemblies, unlike in macro joint counterparts. The risk and severity of voiding in these thinner joints is confirmed to depend on the type and amount of incorporated organic impurities in the electroplated Cu. Therefore, in the present report we show how the bath additive chemistry and various Cu electroplating parameters play a systematic role in the type and quantity of impurity incorporation, and thus the propensity for voiding. Approaches to control the electroplating Cu quality in a way that leads eventually to void-free micro joints are also discussed.

Chromosome-scale genome sequencing, assembly and annotation of six genomes from subfamily Leishmaniinae

We provide the raw and processed data produced during the genome sequencing of isolates from six species of parasites from the sub-family Leishmaniinae: Leishmania martiniquensis (Thailand), Leishmania orientalis (Thailand), Leishmania enriettii (Brazil), Leishmania sp. Ghana, Leishmania sp. Namibia and Porcisia hertigi (Panama). De novo assembly was performed using Nanopore long reads to construct chromosome backbone scaffolds. We then corrected erroneous base calling by mapping short Illumina paired-end reads onto the initial assembly. Data has been deposited at NCBI as follows: raw sequencing output in the Sequence Read Archive, finished genomes in GenBank, and ancillary data in BioSample and BioProject. Derived data such as quality scoring, SAM files, genome annotations and repeat sequence lists have been deposited in Lancaster University’s electronic data archive with DOIs provided for each item. Our coding workflow has been deposited in GitHub and Zenodo repositories. This data constitutes a resource for the comparative genomics of parasites and for further applications in general and clinical parasitology.

Tolerance Design of Multistage Aero-Engine Casing Assembly by Vibration Characteristic Evaluation

A multistage aero-engine casing is a typical thin-walled structural component that is required to maintain a stable dynamic performance because it is a key component for transmitting thrust from an engine to a plane. However, the manufacturing and assembly processes will generate initial assembly deviation, influencing the mechanical properties of the casing during service. Previous tolerance design had few considerations of the effect of assembly deviation on the dynamic characteristic stability. In this paper, a novel performance-oriented tolerance design method for multistage aero-engine casing is proposed. Based on the shell theory, an analytical model is constructed to present the bolted jointed multistage casing assembly. By varying the assembly variables, the mapping relationship between the manufacturing precision and the indeterminacy of dynamic characteristic is established. Through the mapping relationship, an acceptable assembly tolerance with a specified stability requirement is obtained and adopted into the finite element model to show the effectiveness of the actual assembly structure. The presented method provides a significant reference for tolerance design in the actual assembly of the multistage casing.

A multi-step nucleation process determines the kinetics of prion-like domain phase separation

Compartmentalization by liquid-liquid phase separation (LLPS) has emerged as a ubiquitous mechanism underlying the organization of biomolecules in space and time. Here, we combine rapid-mixing time-resolved small-angle X-ray scattering (SAXS) approaches to characterize the assembly kinetics of a prototypical prion-like domain with equilibrium techniques that characterize its phase boundaries and the size distribution of clusters prior to phase separation. We find two kinetic regimes on the micro- to millisecond timescale that are distinguished by the size distribution of clusters. At the nanoscale, small complexes are formed with low affinity. After initial unfavorable complex assembly, additional monomers are added with higher affinity. At the mesoscale, assembly resembles classical homogeneous nucleation. Careful multi-pronged characterization is required for the understanding of condensate assembly mechanisms and will promote understanding of how the kinetics of biological phase separation is encoded in biomolecules.

Structure of the molecular bushing of the bacterial flagellar motor

The basal body of the bacterial flagellum is a rotary motor that consists of several rings (C, MS and LP) and a rod. The LP ring acts as a bushing supporting the distal rod for its rapid and stable rotation without much friction. Here, we use electron cryomicroscopy to describe the LP ring structure around the rod, at 3.5 Å resolution, from Salmonella Typhimurium. The structure shows 26-fold rotational symmetry and intricate intersubunit interactions of each subunit with up to six partners, which explains the structural stability. The inner surface is charged both positively and negatively. Positive charges on the P ring (the part of the LP ring that is embedded within the peptidoglycan layer) presumably play important roles in its initial assembly around the rod with a negatively charged surface.

Pericentromeric chromatin reorganisation follows the initiation of recombination and coincides with early events of synapsis in cereals.

The reciprocal exchange of genetic information between homologous chromosomes during meiotic recombination is essential to secure balanced chromosome segregation and to promote genetic diversity. The chromosomal position and frequency of reciprocal genetic exchange shapes the efficiency of breeding programmes and influences crop improvement under a changing climate. In large genome cereals, such as wheat and barley, crossovers are consistently restricted to subtelomeric chromosomal regions, thus preventing favourable allele combinations being formed within a considerable proportion of the genome, including interstitial and pericentromeric chromatin. Understanding the key elements driving crossover designation is therefore essential to broaden the regions available for crossovers. Here, we followed early meiotic chromatin dynamism in cereals through the visualisation of a homologous barley chromosome arm pair stably transferred into the wheat genetic background. By capturing the dynamics of a single chromosome arm at the same time as detecting the undergoing events of meiotic recombination and synapsis, we showed that subtelomeric chromatin of homologues synchronously transitions to an open chromatin structure during recombination initiation. By contrast, pericentromeric and interstitial regions preserved their closed chromatin organisation and become unpackaged only later, concomitant with initiation of recombinatorial repair and the initial assembly of the synaptonemal complex. Our results raise the possibility that the closed pericentromeric chromatin structure in cereals may influence the fate decision during recombination initiation, as well as the spatial development of synapsis, and may also explain the suppression of crossover events in the proximity of the centromeres.

An exploration of assembly strategies and quality metrics on the accuracy of the rewarewa (Knightia excelsa) genome.

We used long read sequencing data generated from Knightia excelsa, a nectar‐producing Proteaceae tree endemic to Aotearoa (New Zealand), to explore how sequencing data type, volume and workflows can impact final assembly accuracy and chromosome reconstruction. Establishing a high‐quality genome for this species has specific cultural importance to Māori and commercial importance to honey producers in Aotearoa. Assemblies were produced by five long read assemblers using data subsampled based on read lengths, two polishing strategies and two Hi‐C mapping methods. Our results from subsampling the data by read length showed that each assembler tested performed differently depending on the coverage and the read length of the data. Subsampling highlighted that input data with longer read lengths but perhaps lower coverage constructed more contiguous, kmers and gene‐complete assemblies than short read length input data with higher coverage. The final genome assembly was constructed into 14 pseudochromosomes using an initial flye long read assembly, a racon/medaka/pilon combined polishing strategy, salsa2 and allhic scaffolding, juicebox curation, and Macadamia linkage map validation. We highlighted the importance of developing assembly workflows based on the volume and read length of sequencing data and established a robust set of quality metrics for generating high‐quality assemblies. Scaffolding analyses highlighted that problems found in the initial assemblies could not be resolved accurately by Hi‐C data and that assembly scaffolding was more successful when the underlying contig assembly was of higher accuracy. These findings provide insight into how quality assessment tools can be implemented throughout genome assembly pipelines to inform the de novo reconstruction of a high‐quality genome assembly for nonmodel organisms.

Chromosome-level genome assembly of the blue crab, Callinectes sapidus.

The blue crab, Callinectes sapidus (Rathbun, 1896) is an economically, culturally, and ecologically important species found across the temperate and tropical North and South American Atlantic coast. A reference genome will enable research for this high-value species. Initial assembly combined 200× coverage Illumina paired-end reads, a 60× 8 kb mate-paired library, and 50× PacBio data using the MaSuRCA assembler resulting in a 985 Mb assembly with a scaffold N50 of 153 kb. Dovetail Chicago and HiC sequencing with the 3d DNA assembler and Juicebox assembly tools were then used for chromosome scaffolding. The 50 largest scaffolds span 810 Mb are 1.5–37 Mb long and have a repeat content of 36%. The 190 Mb unplaced sequence is in 3921 sequences over 10 kb with a repeat content of 68%. The final assembly N50 is 18.9 Mb for scaffolds and 9317 bases for contigs. Of arthropod BUSCO, ∼88% (888/1013) were complete and single copies. Using 309 million RNAseq read pairs from 12 different tissues and developmental stages, 25,249 protein-coding genes were predicted. Between C. sapidus and Portunus trituberculatus genomes, 41 of 50 large scaffolds had high nucleotide identity and protein-coding synteny, but 9 scaffolds in both assemblies were not clear matches. The protein-coding genes included 9423 one-to-one putative orthologs, of which 7165 were syntenic between the two crab species. Overall, the two crab genome assemblies show strong similarities at the nucleotide, protein, and chromosome level and verify the blue crab genome as an excellent reference for this important seafood species.

Development of Genomic SSR for the Subtropical Hardwood Tree Dalbergia hupeana and Assessment of Their Transferability to Other Related Species

Dalbergia hupeana Hance (D. hupeana) is a precious hardwood tree of the genus Dalbergia. It is one of the few species widely distributed within subtropical areas and is important for timber production and forest restoration. At present, there is little published genetic information on D. hupeana. Therefore, we performed a genome survey using next generation sequencing (NGS) and developed a set of novel genomic SSR (gSSR) markers from the assembled data, and assessed the transferability of these markers to other Dalbergia species in Asia. The results of the genome survey show the genome size of D. hupeana to be about 664 Mb and highly heterozygous. The assembly of sequencing data produced 2,431,997 contigs, and the initial assembly of the NGS data alone resulted in contig N50 of 393 kb with a total of 720 Mb. A total of 127,742 perfect SSR markers were found in the assembled contigs. A total of 37 highly polymorphic and easily genotyped gSSR markers were developed in D. hupeana, while the majority of gSSR markers could be successfully transferred to nine other Dalbergia species in Asia. The transferability rate of gSSR markers was highest in D. balansae, which is more closely related to D. hupeana. Seven gSSR markers were able to be amplified in all tested species. In addition, a preliminary assessment of the genetic diversity of three tree species in the Dalbergia genus suggested a high level of genetic diversity within populations distributed in the subtropical area in China. However, the determination of the global status of their genetic variation still requires further and more comprehensive assessment. Our findings will enable further studies on the genetic diversity, phylogenetics, germplasm characterization, and taxonomy of various Dalbergia species.

MoSep3 and MoExo70 are needed for MoCK2 ring assembly essential for appressorium function in the rice blast fungus, Magnaporthe oryzae.

Polar growth during appressorium formation is vital for the penetration peg formation in the rice blast fungus, Magnaporthe oryzae. Previous research has shown that the Sln1‐septin‐exocyst complex, localized at the base of the appressorium in contact with the leaf surface, forms a ring structure that influences growth polarity and affects penetration peg formation, and is necessary for pathogenicity. Our previous research showed CK2 proteins assemble another ring structure positioned perpendicular to the Sln1‐septin‐exocyst complex. Our research showed that the CK2 ring needs to become correctly assembled for penetration peg function and subsequent plant infection. In the present study, we found that the ring structures of CK2 are absent in the appressorium of ΔMoSep3 septin deletion mutants lacking the septin ring of the Sln1‐septin‐exocyst complex. Sln1 affects the septin proteins that recruit the exocyst complex that localizes as another ring at the appressorium's bottom. Destruction of the exocyst complex by mutation also causes incorrect localization of the CK2 ring structure. In conclusion, CK2 probably takes part in reestablishing the appressorium' spolarity growth necessary for penetration peg formation. We can also conclude that the correct localization and assembly of one or more CK2 ring structures in the appressorium depend on the initial assembly of the Sln1‐septin‐exocyst complex two rings at the base of the appressorium.

Assembly of the Populus Microbiome Is Temporally Dynamic and Determined by Selective and Stochastic Factors.

ABSTRACTRecent work shows that the plant microbiome, particularly the initial assembly of this microbiome, influences plant health, survival, and fitness. Here, we characterize the initial assembly of the Populus microbiome across ten genotypes belonging to two poplar species in a common garden using 16S rRNA gene and ITS2 region amplicon sequencing of the leaf endosphere, leaf surface, root endosphere, and rhizosphere. We sampled these microbiomes three times throughout the first growing season and found that the composition of the microbiome changed dramatically over time across all plant-associated habitats and host genotypes. For archaea and bacteria, these changes were dominated by strong homogenizing selection (accounting for 29 to 62% of pairwise comparisons). However, fungal assembly was generally characterized by multiple ecological assembly processes (i.e., a mix of weak selective and dispersal processes). Interestingly, genotype, while a significant moderator of microbiome composition, generally explained less variation than sample date across plant-associated habitats. We defined a set of core genera that accounted for, on average, 36% of the microbiome. The relative abundance of this core community was consistent over time. Additionally, using source tracking modeling, we determined that new microbial taxa colonize from both aboveground and belowground sources, and combined with our ecological assembly null models, we found that both selective and dispersal processes explained the differences between exo- (i.e., leaf surface and rhizosphere) and endospheric microbiomes. Taken together, our results suggest that the initial assembly of the Populus microbiome is time-, genotype-, and habitat-dependent and is moderated by both selective and stochastic factors.IMPORTANCE The initial assembly of the plant microbiome may establish the trajectory of forthcoming microbiome states, which could determine the overall future health of the plant. However, while much is known about the initial microbiome assembly of grasses and agricultural crops, less is known about the initial microbiome of long-lived trees, such as poplar (Populus spp.). Thus, a greater understanding of initial plant microbiome assembly in an ecologically and economically important plant such as Populus is highly desirable. Here, we show that the initial microbiome community composition and assembly in the first growing season of Populus is temporally dynamic and is determined by a combination of both selective and stochastic factors. Our findings could be used to prescribe ecologically informed microbial inoculations and better predict the composition of the Populus microbiome into the future and to better understand its influence on plant health.

Control of the Serine Integrase Reaction: Roles of the Coiled-Coil and Helix E Regions in DNA Site Synapsis and Recombination.

Bacteriophage serine integrases catalyze highly specific recombination reactions between defined DNA segments called att sites. These reactions are reversible depending upon the presence of a second phage-encoded directionality factor. The bipartite C-terminal DNA-binding region of integrases includes a recombinase domain (RD) connected to a zinc-binding domain (ZD), which contains a long flexible coiled-coil (CC) motif that extends away from the bound DNA. We directly show that the identities of the phage A118 integrase att sites are specified by the DNA spacing between the RD and ZD DNA recognition determinants, which in turn directs the relative trajectories of the CC motifs on each subunit of the att-bound integrase dimer. Recombination between compatible dimer-bound att sites requires minimal-length CC motifs and 14 residues surrounding the tip where the pairing of CC motifs between synapsing dimers occurs. Our alanine-scanning data suggest that molecular interactions between CC motif tips may differ in integrative (attP × attB) and excisive (attL × attR) recombination reactions. We identify mutations in 5 residues within the integrase oligomerization helix that control the remodeling of dimers into tetramers during synaptic complex formation. Whereas most of these gain-of-function mutants still require the CC motifs for synapsis, one mutant efficiently, but indiscriminately, forms synaptic complexes without the CC motifs. However, the CC motifs are still required for recombination, suggesting a function for the CC motifs after the initial assembly of the integrase synaptic tetramer. IMPORTANCE The robust and exquisitely regulated site-specific recombination reactions promoted by serine integrases are integral to the life cycle of temperate bacteriophage and, in the case of the A118 prophage, are an important virulence factor of Listeria monocytogenes. The properties of these recombinases have led to their repurposing into tools for genetic engineering and synthetic biology. In this report, we identify determinants regulating synaptic complex formation between correct DNA sites, including the DNA architecture responsible for specifying the identity of recombination sites, features of the unique coiled-coil structure on the integrase that are required to initiate synapsis, and amino acid residues on the integrase oligomerization helix that control the remodeling of synapsing dimers into a tetramer active for DNA strand exchange.

Dog10K_Boxer_Tasha_1.0: A Long-Read Assembly of the Dog Reference Genome.

The domestic dog has evolved to be an important biomedical model for studies regarding the genetic basis of disease, morphology and behavior. Genetic studies in the dog have relied on a draft reference genome of a purebred female boxer dog named “Tasha” initially published in 2005. Derived from a Sanger whole genome shotgun sequencing approach coupled with limited clone-based sequencing, the initial assembly and subsequent updates have served as the predominant resource for canine genetics for 15 years. While the initial assembly produced a good-quality draft, as with all assemblies produced at the time, it contained gaps, assembly errors and missing sequences, particularly in GC-rich regions, which are found at many promoters and in the first exons of protein-coding genes. Here, we present Dog10K_Boxer_Tasha_1.0, an improved chromosome-level highly contiguous genome assembly of Tasha created with long-read technologies that increases sequence contiguity >100-fold, closes >23,000 gaps of the CanFam3.1 reference assembly and improves gene annotation by identifying >1200 new protein-coding transcripts. The assembly and annotation are available at NCBI under the accession GCF_000002285.5.

Evaluating the accuracy of Listeria monocytogenes assemblies from quasimetagenomic samples using long and short reads

BackgroundWhole genome sequencing of cultured pathogens is the state of the art public health response for the bioinformatic source tracking of illness outbreaks. Quasimetagenomics can substantially reduce the amount of culturing needed before a high quality genome can be recovered. Highly accurate short read data is analyzed for single nucleotide polymorphisms and multi-locus sequence types to differentiate strains but cannot span many genomic repeats, resulting in highly fragmented assemblies. Long reads can span repeats, resulting in much more contiguous assemblies, but have lower accuracy than short reads.ResultsWe evaluated the accuracy of Listeria monocytogenes assemblies from enrichments (quasimetagenomes) of naturally-contaminated ice cream using long read (Oxford Nanopore) and short read (Illumina) sequencing data. Accuracy of ten assembly approaches, over a range of sequencing depths, was evaluated by comparing sequence similarity of genes in assemblies to a complete reference genome. Long read assemblies reconstructed a circularized genome as well as a 71 kbp plasmid after 24 h of enrichment; however, high error rates prevented high fidelity gene assembly, even at 150X depth of coverage. Short read assemblies accurately reconstructed the core genes after 28 h of enrichment but produced highly fragmented genomes. Hybrid approaches demonstrated promising results but had biases based upon the initial assembly strategy. Short read assemblies scaffolded with long reads accurately assembled the core genes after just 24 h of enrichment, but were highly fragmented. Long read assemblies polished with short reads reconstructed a circularized genome and plasmid and assembled all the genes after 24 h enrichment but with less fidelity for the core genes than the short read assemblies.ConclusionThe integration of long and short read sequencing of quasimetagenomes expedited the reconstruction of a high quality pathogen genome compared to either platform alone. A new and more complete level of information about genome structure, gene order and mobile elements can be added to the public health response by incorporating long read analyses with the standard short read WGS outbreak response.

Assembly sequence and path planning for monotone and nonmonotone assemblies with rigid and flexible parts

• SPP-Flex method for assembly sequence and path planning of rigid and flexible parts. • A new greedy assembly sequence planning heuristic minimizes assembly stress on parts. • A new sampling-based path planner (BXXT) is proposed for path planning of parts. • Assembling flexible parts is simulated and analyzed via the Abaque FEA software. • Four variants of SPP-Flex are proposed to solve monotone and nonmonotone products. The Assembly Sequence and Path Planning (ASPP) problem deals with finding a proper sequence of parts to be assembled into a finished product and short assembly paths for each part. The problem is a combination of Assembly Sequence Planning (ASP) and Assembly Path Planning (APP) subproblems, which are both NP-complete and therefore intractable at large sizes. Nearly in all works on ASPP, it is assumed that planning is monotone (i.e., parts are moved only once, without considering intermediate placements) and each part is completely rigid. These are simplifying, yet limiting assumptions, since most assembled products like ships, aircraft, and automobiles are composed of rigid and flexible parts, and the generation of assembly sequence and path plans for most real-world products requires intermediate placement of parts to be taken into account. None of the existing works in the literature, however, have handled nonmonotone ASPP problems for rigid and flexible parts, and this issue remains largely untouched. In this paper, we present a new method called SPP-Flex for solving monotone and nonmonotone ASPP for rigid and flexible parts. SPP-Flex first utilizes a Directional Assembly Stress Matrix (DASM) for describing interference relations between all pairs of parts and the amounts of compressive stresses needed for assembling flexible parts and then obtains an initial tentative assembly sequence using a simple new greedy heuristic. Next, short assembly paths are iteratively computed and planned from initial to goal configurations of all parts using a novel sampling-based path planner called BXXT. If finding a free path for an active part fails due to obstruction of a previously assembled part, then such a part is identified, relocated, and its path replanned until the active part is moved to its final position. In case of failure again, if the part is flexible, through finite element analysis, it is determined if the part can still be assembled by undergoing elastic deformation. To evaluate the performance of the SPP-Flex and its components, two new products were designed and solved by four combinations of ASP and APP methods 20 times each, and the means and standard deviations of five performance criteria (total path length, total number of generated nodes and edges in the search tree, total number of collision (interference) checks, and total runtime) were calculated. Analysis of the computational results showed that the proposed greedy heuristic sequence planner together with the BXXT path planner/replanner outperformed other variations with at most 4.6% average gap in path length and 2.1% average gap in runtime compared to the best-found solution in all runs.

37 Genomic improvement of the horse X chromosome and characterization of the pseudoautosomal boundary

The horse reference genome has been improved with the release of EquCab3 and the first Y chromosome reference. The most complex sequences, such as those in the sex chromosomes, are still unresolved. These include large amplicons, repeats, and the pseudoautosomal boundary (PAB). We initiated a comprehensive study specifically to improve the assembly of the horse sex chromosomes. To refine the assembly of complex regions, we are utilizing 3 new technologies: trio-binning, Hi-C and Bionano optical mapping. Trio-binning uses long read sequences from F1 interspecific hybrids and short reads from parent species. High molecular weight blood DNA was extracted from a female hinny and sequenced on 2 PacBio Sequel cells. Paired-end Illumina reads (150bp) for horse ( Twilight ) and donkey ( Willy ) were obtained from SRA. These sequences as well as the hinny long reads were assembled with trio-binning function of the Canu assembler program. The initial assembly is 2.5 Gb separated into 1,757 contigs with an N50 of 41.5 Mb. We have completed Hi-C sequencing and Bionano optical maps for one thoroughbred stallion ( Bravo ). Both technologies are needed to scaffold the trio-binning Canu assembly. Our initial goal was to use this assembly to better define the PAB in the horse. The PAB demarcates the end of the pseudoautosomal region (PAR) where X-Y recombination stops. Despite the evolutionary and biological importance of the PAB, the region has been characterized at molecular level in only a few species. The PAB of the horse is presently not well defined. Previously, we identified and Sanger sequenced 4 BAC clones - 2 spanning PAB-X and PAB-Y. To identify the PAB of the horse, BAC sequences were aligned to the Y assembly, EquCab3 and a 42 Mb-size contig from trio-binning assembly which corresponds to the short arm of the X. We identified a region on both X and Y where X-Y homology drops from over 97% (PAR) to almost zero, indicative of the PAB. This region corresponds to the location of the XKR3Y gene in the Y but is not well-annotated in the X. We also identified a duplication and an inversion in EquCab3 which was not consistent with the corresponding region in the X-BACs, or the new 42 Mb Xp contig, suggesting a mis-assembly in EquCab3. We believe that these approaches combined will also resolve other complex and repetitive portions in the horse sex chromosomes.

Interplay of four auxiliary factors is required for the assembly of photosystem I reaction center subcomplex.

The photosystem I (PSI) complex consisting of reaction center (RC) subunits, several peripheral subunits and many co-factors, is present in the thylakoid membranes of chloroplasts and cyanobacteria. The assembly of RC subunits (PsaA/B) that bind electron transfer co-factors and antenna pigments is an intricate process, and is mediated by several auxiliary factors such as Ycf3, Y3IP1/CGL59, Ycf4 and Ycf37/PYG7/CGL71. However, their precise molecular mechanisms in RC assembly remain to be addressed. Here we purified four PSI auxiliary factors by affinity chromatography, and characterized co-purified PSI assembly intermediates. We suggest that Ycf3 assists the initial assembly of newly synthesized PsaA/B subunits into an RC subcomplex, while Y3IP1 may be involved in transferring the RC subcomplex from Ycf3 to the Ycf4 module that stabilizes it. CGL71 may form an oligomer that transiently interacts with the PSI RC subcomplex, physically protecting it under oxic conditions until association with the peripheral PSI subunits occurs. Together, our results reveal the interplay among four auxiliary factors required for the stepwise assembly of the PSI RC.

Allele-defined genome reveals biallelic differentiation during cassava evolution

Allele-defined genome reveals biallelic differentiation during cassava evolution