Accurate Assembly Research Articles

AccuVIR: an ACCUrate VIRal genome assembly tool for third-generation sequencing data.

RNA viruses tend to mutate constantly. While many of the variants are neutral, some can lead to higher transmissibility or virulence. Accurate assembly of complete viral genomes enables the identification of underlying variants, which are essential for studying virus evolution and elucidating the relationship between genotypes and virus properties. Recently, third-generation sequencing platforms such as Nanopore sequencers have been used for real-time virus sequencing for Ebola, Zika, coronavirus disease 2019, etc. However, their high per-base error rate prevents the accurate reconstruction of the viral genome. In this work, we introduce a new tool, AccuVIR, for viral genome assembly and polishing using error-prone long reads. It can better distinguish sequencing errors from true variants based on the key observation that sequencing errors can disrupt the gene structures of viruses, which usually have a high density of coding regions. Our experimental results on both simulated and real third-generation sequencing data demonstrated its superior performance on generating more accurate viral genomes than generic assembly or polish tools. The source code and the documentation of AccuVIR are available at https://github.com/rainyrubyzhou/AccuVIR. Supplementary data are available at Bioinformatics online.

Reference-based read clustering improves the de novo genome assembly of microbial strains

Constructing accurate microbial genome assemblies is necessary to understand genetic diversity in microbial genomes and its functional consequences. However, it still remains as a challenging task especially when only short-read sequencing technologies are used. Here, we present a new read-clustering algorithm, called RBRC, for improving de novo microbial genome assembly, by accurately estimating read proximity using multiple reference genomes. The performance of RBRC was confirmed by simulation-based evaluation in terms of assembly contiguity and the number of misassemblies, and was successfully applied to existing fungal and bacterial genomes by improving the quality of the assemblies without using additional sequencing data. RBRC is a very useful read-clustering algorithm that can be used (i) for generating high-quality genome assemblies of microbial strains when genome assemblies of related strains are available, and (ii) for upgrading existing microbial genome assemblies when the generation of additional sequencing data, such as long reads, is difficult.

NPGREAT: assembly of human subtelomere regions with the use of ultralong nanopore reads and linked-reads

BackgroundHuman subtelomeric DNA regulates the length and stability of adjacent telomeres that are critical for cellular function, and contains many gene/pseudogene families. Large evolutionarily recent segmental duplications and associated structural variation in human subtelomeres has made complete sequencing and assembly of these regions difficult to impossible for many loci, complicating or precluding a wide range of genetic analyses to investigate their function.ResultsWe present a hybrid assembly method, NanoPore Guided REgional Assembly Tool (NPGREAT), which combines Linked-Read data with mapped ultralong nanopore reads spanning subtelomeric segmental duplications to potentially overcome these difficulties. Linked-Read sets of DNA sequences identified by matches with 1-copy subtelomere sequence adjacent to segmental duplications are assembled and extended into the segmental duplication regions using Regional Extension of Assemblies using Linked-Reads (REXTAL). Mapped telomere-containing ultralong nanopore reads are then used to provide contiguity and correct orientation for matching REXTAL sequence contigs as well as identification/correction of any misassemblies. Our method was tested for a subset of representative subtelomeres with ultralong nanopore read coverage in the haploid human cell line CHM13. A 10X Linked-Read dataset from CHM13 was combined with ultralong nanopore reads from the same genome to provide improved subtelomere assemblies. Comparison of Nanopore-only assemblies using SHASTA with our NPGREAT assemblies in the distal-most subtelomere regions showed that NPGREAT produced higher-quality and more complete assemblies than SHASTA alone when these regions had low ultralong nanopore coverage (such as cases where large segmental duplications were immediately adjacent to (TTAGGG) tracts).ConclusionIn genomic regions with large segmental duplications adjacent to telomeres, NPGREAT offers an alternative economical approach to improving assembly accuracy and coverage using linked-read datasets when more expensive HiFi datasets of 10–20 kb reads are unavailable.

Research on size coupling and sensitivity analysis of SAR antenna truss rod

To eliminate the influence of the size coupling of SAR antenna truss rod on assembly accuracy, this paper defines the assembly sequence, part datum, tolerance, assembly operation, and measurement operation of the model by establishing the tolerance analysis model of the assembly dimension chain and measurement target. The assembly simulation process of the deployment mechanism is simulated, the plane deviation of the antenna panel of the deployment mechanism is analyzed, and the contribution and sensitivity data of each component ring size to the closed ring size are obtained. The simulation results show that the in-plane angle error of the antenna panel is mainly affected by the length of the component ring rod. By focusing on controlling the dimensional tolerance of the truss rods with high sensitivity, the assembly accuracy of the antenna deployment system can be guaranteed. Sensitivity is the magnification of assembly deviation by the transmission of deviation source, which reflects the influence of the change of composition tolerance on the change of functional requirements. According to the sensitivity analysis, the key dimensions that affect assembly accuracy can be obtained. The assembly requirements of high-quality and low-cost products can be achieved by strictly controlling the dimensional tolerance of key components and relaxing the tolerance of non-key components.

Missing Detection of The Clutch-Driven Disc Spring Assembly

A detection system for the missing assembly of clutch-driven plate springs is established. The algorithms used in this system, such as point cloud data preprocessing, spring area positioning, and spring missing detection, are studied. Firstly, according to the characteristics of the clutch-driven plate spring point cloud, the Gaussian statistical method is used to eliminate the outliers incurred by system error and other reasons, and the two-sided filtering is performed on the clutch-driven plate spring point cloud that eliminates the outliers to eliminate the noise points with small ups and downs. Then, the theoretical division area is constructed by using the theoretical width between the clutch-driven plate springs and the theoretical width of the springs. Compared with actually recorded positions, the theoretical area is expanded so that the real spring point cloud data are in the corresponding positioning area; finally, the spring in the region is identified by using the characteristics of an inconsistent number of point clouds in different assembly conditions, so as to detect the missing assembly of the clutch-driven plate spring. The experimental results show that the detection accuracy of the clutch-driven plate spring assembly is as high as 100%. It can basically meet the detection requirements for the missing assembly of clutch-driven plate springs.

Sequence-specific remodeling of a topologically complex RNP substrate by Spb4.

DEAD-box ATPases are ubiquitous enzymes essential in all aspects of RNA biology. However, the limited in vitro catalytic activities described for these enzymes are at odds with their complex cellular roles, most notably in driving large-scale RNA remodeling steps during the assembly of ribonucleoproteins (RNPs). We describe cryo-EM structures of 60S ribosomal biogenesis intermediates that reveal how context-specific RNA unwinding by the DEAD-box ATPase Spb4 results in extensive, sequence-specific remodeling of rRNA secondary structure. Multiple cis and trans interactions stabilize Spb4 in a post-catalytic, high-energy intermediate that drives the organization of the three-way junction at the base of rRNA domain IV. This mechanism explains how limited strand separation by DEAD-box ATPases is leveraged to provide non-equilibrium directionality and ensure efficient and accurate RNP assembly.

In-built resonant structures for precision assembly of X-Ray Optics for 4th Generation Light Sources

As the Diamond Light Source embraces the move towards becoming a fourth generation light source it will require its optics to perform under increasingly demanding conditions. Foremost amongst these conditions will be the increasing powers they are subjected to and the reducing real estate they must perform in. With these new challenges comes the need for greater understanding of how optics are assembled and how consistently this activity is carried out. In this paper, the concept of using passive resonant structures as part of a precision assembly approach for such optics is introduced. Numerical simulation of a passive resonant device comprising tines which are compressed as clamping force is increased is presented, revealing a frequency shift from intermediately fastened to fully tightened. This approach has the potential to provide a robust, game changing improvement to the accuracy of assembly of X-ray optics and subsequently a significant improvement in their performance.

Research on factors affecting the assembly accuracy of deployable arms

A spacecraft deployable arm is one of the key components of spacecraft. The assembly accuracy of space deployable arms restricts the improvement of spacecraft performance. Therefore, it is important to analyze the factors influencing the assembly accuracy of deployable arms to optimize the assembly process. This paper studies the precision assembly of deployable arms, with a spacecraft deployable arm as the specific research object. In the study, the assembly process is modeled theoretically and analyzed by Finite Element Analysis. Finally, the assembly experiment of the deployable arm is carried out based on the research.

Measurement and simulation of thermal-induced residual stresses within friction stir lapped Al/steel plate

Welding residual stress and distortion usually affect the assembly accuracy, product performance, and service life, with no exception for the Al/steel hybrid structure. The lapped joint of 5052 Al alloy and DP590 steel was made by friction stir welding (FSW) at various rotation speeds. A subsequentially coupled computational modeling approach based on the in-house software JWRIAN combining an efficient hybrid solution method was developed to predict the temperature field, the residual stress distribution and the warpage deformation. Based on the simulation, a higher welding temperature with subsequently a higher average cooling rate was created at a high rotation velocity, leading to the area extension of the longitudinal tensile stress and transverse compressive stress near the weld zone. The thermal expansion coefficient mismatch leads to the mirroring distribution of the near interfacial residual stress on both sides, particularly for transverse residual stress. With the increasing rotation speed, the longitudinal residual stress on the steel side near the interface was changed from compressive to the tensile state. The maximum warpage deformation in the Al plate border became larger at a higher rotation speed. The surface residual stress was measured by X-ray diffusion method and the internal residual stress was reconstructed by the contour method with the aid of elastic FE analysis. The comparisons of temperature history, residual stress and deformation indicated satisfactory agreements between simulation and experiment ones.

Practical realization and design of three-cascade prism mass analyzer for isotopic analysis of H-He mixtures using MS-platform technology

The paper presents the construction of a mass analyzer for the analysis of hydrogen-helium mixtures based on a magnetic prism using a three-stage circuit that includes two cylindrical capacitors. The choice of an ion-optical scheme that provides a high resolution of 3300, which is necessary for the separation of all nuclides formed during the analysis of hydrogen-helium mixtures, is justified. It is shown that the innovative «MS-Platform» technology makes it possible to ensure high assembly accuracy of the elements of the prism mass analyzer ion-optical circuit, which is necessary to realize all the advantages of a prism mass spectrometer compared to sector ones.

Rapid and Accurate Assembly of Large DNA Assisted by In Vitro Packaging of Bacteriophage.

Development of DNA assembly methods made it possible to construct large DNA. However, achieving a large DNA assembly easily, accurately, and at a low cost remains a challenge. This study shows that DNA assembled only by annealing of overlapping single-stranded DNA ends, which are generated by exonuclease treatment, without ligation can be packaged in phage particles and can also be transduced into bacterial cells. Based on this, I developed a simple method to construct long DNA of about 40-50 kb from five to ten PCR fragments using the bacteriophage in vitro packaging system. This method, namely, iPac (in vitro Packaging-assisted DNA assembly), allowed accurate and rapid construction of large plasmids and phage genomes. This simple method will accelerate research in molecular and synthetic biology, including the construction of gene circuits or the engineering of metabolic pathways.

Study on the Extrusion Molding Process of Polylactic Acid Micro Tubes for Biodegradable Vascular Stents.

Polylactic acid (PLA) has been widely used in the field of medical devices. However, few studies have been conducted on the extrusion molding of PLA micro tubes for the preparation of biodegradable vascular stents. In this paper, the extrusion die for PLA single-cavity micro tubes was designed and manufactured by micro-extrusion theory. Taking the outer diameter, wall thickness, wall thickness uniformity and ovality of micro tubes as the evaluation index, the influence of the main extrusion process parameters on the evaluation index was studied. The experimental results show that the outer diameter and wall thickness are significantly affected by screw speed, pulling speed and gas flow rate; extrusion process parameters have little influence on wall thickness uniformity and ovality within a certain range, which mainly depends on the processing accuracy and assembly accuracy of the extrusion die. However, excessively high screw speed and low gas flow rate have significant effects on ovality. Finally, according to the influence of extrusion process parameters on the evaluation index, a series of micro tubes that meet the design requirements are extruded and carved into vascular stent structures.

GAVISUNK: genome assembly validation via inter-SUNK distances in Oxford Nanopore reads.

Highly contiguous de novo phased diploid genome assemblies are now feasible for large numbers of species and individuals. Methods are needed to validate assembly accuracy and detect misassemblies with orthologous sequencing data to allow for confident downstream analyses. We developed GAVISUNK, an open-source pipeline that detects misassemblies and produces a set of reliable regions genome-wide by assessing concordance of distances between unique k-mers in Pacific Biosciences high-fidelity assemblies and raw Oxford Nanopore Technologies reads. GAVISUNK is available at https://github.com/pdishuck/GAVISUNK. Supplementary data are available at Bioinformatics online.

The protein common assembly database (ProtCAD)-a comprehensive structural resource of protein complexes.

Proteins often act through oligomeric interactions with other proteins. X-ray crystallography and cryo-electron microscopy provide detailed information on the structures of biological assemblies, defined as the most likely biologically relevant structures derived from experimental data. In crystal structures, the most relevant assembly may be ambiguously determined, since multiple assemblies observed in the crystal lattice may be plausible. It is estimated that 10-15% of PDB entries may have incorrect or ambiguous assembly annotations. Accurate assemblies are required for understanding functional data and training of deep learning methods for predicting assembly structures. As with any other kind of biological data, replication via multiple independent experiments provides important validation for the determination of biological assembly structures. Here we present the Protein Common Assembly Database (ProtCAD), which presents clusters of protein assembly structures observed in independent structure determinations of homologous proteins in the Protein Data Bank (PDB). ProtCAD is searchable by PDB entry, UniProt identifiers, or Pfam domain designations and provides downloads of coordinate files, PyMol scripts, and publicly available assembly annotations for each cluster of assemblies. About 60% of PDB entries contain assemblies in clusters of at least 2 independent experiments. All clusters and coordinates are available on ProtCAD web site (http://dunbrack2.fccc.edu/protcad).

PROJECT CONCEPTION OF CAE-SYSTEM FOR WEAR ANALYSIS OF FRICTION BEARING UNDER CONDITIONS OF SHAFT AND LINER AXIS MISALIGNMENT

Modelling of wear processes is an important stage in the design of mechanisms. Such virtual studies allow predicting the service life of components and the machine as a whole. Modern algorithms of CAE-systems allow performing such analysis with high accuracy and taking into account not only material and operating conditions of the mechanism, but also errors that occurred during the manufacturing and assembly of a particular part. These parts require high requirements to the accuracy of manufacturing and assembly, but within the tolerances of the fields errors of mutual positioning are still allowed, which in turn can significantly affect the service life of the mechanism. Therefore, the task of modeling the wear process of the shaft and sleeve in conditions of mutual misalignment of the axes is relevant. Such sliding pairs exist in almost all machines, including car engines, power generation turbines, etc. In this article it is offered to use the module-integrated approach for modeling of wear processes in tribotechnics, providing use of CAE-systems for definition of functioning conditions of friction units as a system core, and construction of separate modules which in this or that degree are integrated into existing CAE systems. to define values of wear of elements of friction pairs that allows to consider mutual influence of wear and change of functioning conditions in the course of operation. The practical implementation of the module-integrated approach is shown by the example of solving the problem of radial plain bearings wear during the misalignment of the shaft and bush axes in the ANSYS environment. The module of representation of multidimensional matrixes of static or dynamic data, providing essential increase in speed of data processing and quality level of the further analysis of the visualized information from any subject area, is developed.

Architectural groups of a subtelomeric gene family evolve along distinct paths in Candida albicans.

Subtelomeres are dynamic genomic regions shaped by elevated rates of recombination, mutation, and gene birth/death. These processes contribute to formation of lineage-specific gene family expansions that commonly occupy subtelomeres across eukaryotes. Investigating the evolution of subtelomeric gene families is complicated by the presence of repetitive DNA and high sequence similarity among gene family members that prevents accurate assembly from whole genome sequences. Here, we investigated the evolution of the telomere-associated (TLO) gene family in Candida albicans using 189 complete coding sequences retrieved from 23 genetically diverse strains across the species. Tlo genes conformed to the 3 major architectural groups (α/β/γ) previously defined in the genome reference strain but significantly differed in the degree of within-group diversity. One group, Tloβ, was always found at the same chromosome arm with strong sequence similarity among all strains. In contrast, diverse Tloα sequences have proliferated among chromosome arms. Tloγ genes formed 7 primary clades that included each of the previously identified Tloγ genes from the genome reference strain with 3 Tloγ genes always found on the same chromosome arm among strains. Architectural groups displayed regions of high conservation that resolved newly identified functional motifs, providing insight into potential regulatory mechanisms that distinguish groups. Thus, by resolving intraspecies subtelomeric gene variation, it is possible to identify previously unknown gene family complexity that may underpin adaptive functional variation.

Semi-automated assembly of high-quality diploid human reference genomes

The current human reference genome, GRCh38, represents over 20 years of effort to generate a high-quality assembly, which has benefitted society1,2. However, it still has many gaps and errors, and does not represent a biological genome as it is a blend of multiple individuals3,4. Recently, a high-quality telomere-to-telomere reference, CHM13, was generated with the latest long-read technologies, but it was derived from a hydatidiform mole cell line with a nearly homozygous genome5. To address these limitations, the Human Pangenome Reference Consortium formed with the goal of creating high-quality, cost-effective, diploid genome assemblies for a pangenome reference that represents human genetic diversity6. Here, in our first scientific report, we determined which combination of current genome sequencing and assembly approaches yield the most complete and accurate diploid genome assembly with minimal manual curation. Approaches that used highly accurate long reads and parent–child data with graph-based haplotype phasing during assembly outperformed those that did not. Developing a combination of the top-performing methods, we generated our first high-quality diploid reference assembly, containing only approximately four gaps per chromosome on average, with most chromosomes within ±1% of the length of CHM13. Nearly 48% of protein-coding genes have non-synonymous amino acid changes between haplotypes, and centromeric regions showed the highest diversity. Our findings serve as a foundation for assembling near-complete diploid human genomes at scale for a pangenome reference to capture global genetic variation from single nucleotides to structural rearrangements.

Double-spherically bent crystal scheme of stigmatic x ray monochromatic backlit imaging.

We propose an aberration-free monochromatic x ray backlit imaging scheme using a combination of convex and concave spherically bent crystals. This configuration works with a wide range of Bragg angles, satisfying the conditions for stigmatic imaging at a particular wavelength. However, the assembly accuracy of the crystals must meet the Bragg relation criteria for spatial resolution to increase the detection efficiency. Here, we develop a collimator prism with a cross reference line engraved on a plane mirror to adjust a matched pair of Bragg angles as well as the intervals between the two crystals and the object to be coupled with the detector. We explore the realization of monochromatic backlighting imaging with a concave Si-533 crystal and a convex α-Quartz-2023 crystal, obtaining a spatial resolution of approximately 7 µm and a field of view of at least 200 µm. To the best of our knowledge, this is the best spatial resolution of monochromatic images of a double-spherically bent crystal to date. Our experimental results are presented to demonstrate the feasibility of this imaging scheme with x rays.

Iteratively mapping ancient DNA to reconstruct highly divergent mitochondrial genomes: An evaluation of software, parameters and bait reference

Abstract The ability to obtain ancient DNA (aDNA) from extinct species has greatly improved our ability to study their evolutionary histories. Most aDNA studies focus on mitochondrial DNA for both population‐level and species‐level insights, due to its relatively high abundance and increased obtainability. Although several approaches are available to assemble mitogenomes, high levels of DNA damage and non‐target contaminating DNA make many approaches unsuitable for aDNA. Further complicating matters for most extinct species is the lack of a sufficiently closely related extant relative to act as a reference. Iterative mapping can assemble mitogenomes without using a close reference sequence, and has been applied to aDNA from various extinct species. Despite its widespread use, the accuracy of the reconstructed assemblies is yet to be comprehensively assessed. Here, we investigated the influence of (i) mapping software (Burrows–Wheeler Alignment [BWA], MITObim and NOVOplasty), (ii) parameters and (iii) bait reference phylogenetic distance on assembly accuracy using two simulated datasets: spotted hyena and various mammalian bait references; southern cassowary and various avian bait references, and two empirical datasets: extinct cave hyena; two extinct sloth species. Specifically, we assessed the accuracy of results through three parameters: pairwise distance to a reference conspecific mitogenome, number of incorrectly inserted base pairs and total length of the assembly. We found large discrepancies in the accuracy of reconstructed assemblies using different mapping software, parameters and bait references. Results also varied depending on the dataset, thereby making broad, generalised conclusions difficult. However, the overall most accurate results were obtained with either BWA using a minimum mapping quality of 20 and relaxed mismatch parameters (‐n 0.001 ‐o 2), or MITObim using a mismatch value of 3 or 5. We also show accuracy could be further improved by combining results from multiple bait references. Our study provides information on how divergent mitogenomes are best reconstructed from aDNA. By obtaining the most accurate reconstruction possible, one can be more confident as to the reliability of downstream analyses, and the evolutionary inferences made from them.

The Sequence Dependent Nanoscale Structure of CENP-A Nucleosomes.

CENP-A is a histone variant found in high abundance at the centromere in humans. At the centromere, this histone variant replaces the histone H3 found throughout the bulk chromatin. Additionally, the centromere comprises tandem repeats of α-satellite DNA, which CENP-A nucleosomes assemble upon. However, the effect of the DNA sequence on the nucleosome assembly and centromere formation remains poorly understood. Here, we investigated the structure of nucleosomes assembled with the CENP-A variant using Atomic Force Microscopy. We assembled both CENP-A nucleosomes and H3 nucleosomes on a DNA substrate containing an α-satellite motif and characterized their positioning and wrapping efficiency. We also studied CENP-A nucleosomes on the 601-positioning motif and non-specific DNA to compare their relative positioning and stability. CENP-A nucleosomes assembled on α-satellite DNA did not show any positional preference along the substrate, which is similar to both H3 nucleosomes and CENP-A nucleosomes on non-specific DNA. The range of nucleosome wrapping efficiency was narrower on α-satellite DNA compared with non-specific DNA, suggesting a more stable complex. These findings indicate that DNA sequence and histone composition may be two of many factors required for accurate centromere assembly.