Fragment Assembly Research Articles

Experimental Evaluation of Genetic Algorithms to Solve the DNA Assembly Optimization Problem

This paper aims to highlight the motivations for investigating genetic algorithms (GAs) to solve the DNA Fragment Assembly (DNAFA) problem. DNAFA problem is an optimization problem that attempts to reconstruct an original DNA sequence by finding the shortest DNA sequence from a given set of fragments. This paper is a continuation of our previous research paper in which the existence of a polynomial-time reduction of DNAFA into the Traveling Salesman Problem (TSP) and the Quadratic Assignment Problem (QAP) was discussed. Taking advantage of this reduction, this work conceptually designed a genetic algorithm (GA) platform to solve the DNAFA problem. This platform offers several ingredients enabling us to create several variants of GA solvers for the DNAFA optimization problems. The main contribution of this paper is the designing of an efficient GA variant by carefully integrating different GAs operators of the platform. For that, this work individually studied the effects of different GAs operators on the performance of solving the DNAFA problem. This study has the advantage of benefiting from prior knowledge of the performance of these operators in the contexts of the TSP and QAP problems. The best designed GA variant shows a significant improvement in accuracy (overlap score) reaching more than 172% of what is reported in the literature.

Synthesis and assembly of full-length cyanophage A-4L genome

Artificial cyanophages are considered to be an effective biological method to control harmful cyanobacterial bloom. However, no synthetic cyanophage genome has been constructed and where its obstacles are unclear. Here, we survey a stretch of 16 kb length sequence of cyanophage A-4L that is unclonable in Escherichia coli. We test 12 predicted promoters of cyanophage A-4L which were verified all active in E. coli. Next, we screen for eight ORFs that hindered the assembly of intermediate DNA fragments in E. coli and describe that seven ORFs in the 16 kb sequence could not be separately cloned in E. coli. All of unclonable ORFs in high-copy-number plasmid were successfully cloned using low-copy-number vector, suggesting that these ORFs were copy-number-dependent. We propose a clone strategy abandoned the promotor and the start codon that could be applied for unclonable ORFs. Last, we de novo synthesized and assembled the full-length genome of cyanophage A-4L. This work deepens the understanding of synthetic cyanophages studies.

A Peptides Prediction Methodology with Fragments and CNN for Tertiary Structure Based on GRSA2

Proteins are macromolecules essential for living organisms. However, to perform their function, proteins need to achieve their Native Structure (NS). The NS is reached fast in nature. By contrast, in silico, it is obtained by solving the Protein Folding problem (PFP) which currently has a long execution time. PFP is computationally an NP-hard problem and is considered one of the biggest current challenges. There are several methods following different strategies for solving PFP. The most successful combine computational methods and biological information: I-TASSER, Rosetta (Robetta server), AlphaFold2 (CASP14 Champion), QUARK, PEP-FOLD3, TopModel, and GRSA2-SSP. The first three named methods obtained the highest quality at CASP events, and all apply the Simulated Annealing or Monte Carlo method, Neural Network, and fragments assembly methodologies. In the present work, we propose the GRSA2-FCNN methodology, which assembles fragments applied to peptides and is based on the GRSA2 and Convolutional Neural Networks (CNN). We compare GRSA2-FCNN with the best state-of-the-art algorithms for PFP, such as I-TASSER, Rosetta, AlphaFold2, QUARK, PEP-FOLD3, TopModel, and GRSA2-SSP. Our methodology is applied to a dataset of 60 peptides and achieves the best performance of all methods tested based on the common metrics TM-score, RMSD, and GDT-TS of the area.

Chromosome-level assembly of the Phytophthora agathidicida genome reveals adaptation in effector gene families.

Phytophthora species are notorious plant pathogens, with some causing devastating tree diseases that threaten the survival of their host species. One such example is Phytophthora agathidicida, the causal agent of kauri dieback - a root and trunk rot disease that kills the ancient, iconic and culturally significant tree species, Agathis australis (New Zealand kauri). A deeper understanding of how Phytophthora pathogens infect their hosts and cause disease is critical for the development of effective treatments. Such an understanding can be gained by interrogating pathogen genomes for effector genes, which are involved in virulence or pathogenicity. Although genome sequencing has become more affordable, the complete assembly of Phytophthora genomes has been problematic, particularly for those with a high abundance of repetitive sequences. Therefore, effector genes located in repetitive regions could be truncated or missed in a fragmented genome assembly. Using a combination of long-read PacBio sequences, chromatin conformation capture (Hi-C) and Illumina short reads, we assembled the P. agathidicida genome into ten complete chromosomes, with a genome size of 57 Mb including 34% repeats. This is the first Phytophthora genome assembled to chromosome level and it reveals a high level of syntenic conservation with the complete genome of Peronospora effusa, the only other completely assembled genome sequence of an oomycete. All P. agathidicida chromosomes have clearly defined centromeres and contain candidate effector genes such as RXLRs and CRNs, but in different proportions, reflecting the presence of gene family clusters. Candidate effector genes are predominantly found in gene-poor, repeat-rich regions of the genome, and in some cases showed a high degree of duplication. Analysis of candidate RXLR effector genes that occur in multicopy gene families indicated half of them were not expressed in planta. Candidate CRN effector gene families showed evidence of transposon-mediated recombination leading to new combinations of protein domains, both within and between chromosomes. Further analysis of this complete genome assembly will help inform new methods of disease control against P. agathidicida and other Phytophthora species, ultimately helping decipher how Phytophthora pathogens have evolved to shape their effector repertoires and how they might adapt in the future.

An Optimized Transformation Protocol for Escherichia coli BW3KD with Supreme DNA Assembly Efficiency.

DNA cloning requires two steps: the assembly of recombinant DNA molecules and the transformation of the product into a host organism for replication. High efficiencies in both processes can increase the success rate. Recently, we developed an Escherichia coli BW3KD strain with higher transformation efficiency than commonly used cloning strains. Here, we further developed a simple method named TSS-HI (transformation storage solution optimized by Hannahan and Inoue method) for competent cell preparation, which combined the advantages of three common methods for operational simplicity and high transformation efficiency. When competent BW3KD cells were prepared using this developed method, the transformation efficiency reached up to (7.21 ± 1.85) × 109 CFU/μg DNA, which exceeded the levels of commercial chemically competent cells and homemade electrocompetent cells. BW3KD cells formed colonies within 7 h on lysogeny broth agar plates, quicker than the well-known fast-growing E. coli cloning strain Mach1. The competent cells worked effectively for the transformation of assembled DNA of 1 to 7 fragments in one step and promoted efficiencies of transformation or cloning with large plasmids. The cloning efficiency of BW3KD cells prepared by this method increased up to 828-fold over that of E. coli XL1-Blue MRF' cells prepared by a common method. Thus, competent cells are suitable for different cloning jobs and should help with the increased demand for DNA assembly in biological studies and biotechnology. IMPORTANCE DNA transformation is commonly used in cloning; however, high transformation efficiency becomes a limiting factor in many applications, such as the construction of CRISPR and DNA libraries, the assembly of multiple fragments, and the transformation of large plasmids. We developed a new competent cell preparation method with unmatched transformation efficiency. When the BW3KD strain, derived from Escherichia coli BW25113 cells, was prepared by this method, its transformation efficiency reached up to (7.21 ± 1.85) × 109 CFU/μg DNA, which broke the record for chemically prepared competent cells. Routine cloning could be completed in 1 day due to the high growth rate of this strain. The competent cells were shown to be highly efficient for transformation or cloning with large plasmids and for the assembly of multiple fragments. The results highlight the effectiveness of the new protocol and the usefulness of the BW3KD strain as the host.

ACtivE: Assembly and CRISPR-Targeted in Vivo Editing for Yeast Genome Engineering Using Minimum Reagents and Time.

Thanks to its sophistication, the CRISPR/Cas system has been a widely used yeast genome editing method. However, CRISPR methods generally rely on preassembled DNAs and extra cloning steps to deliver gRNA, Cas protein, and donor DNA. These laborious steps might hinder its usefulness. Here, we propose an alternative method, Assembly and CRISPR-targeted in vivo Editing (ACtivE), that only relies on in vivo assembly of linear DNA fragments for plasmid and donor DNA construction. Thus, depending on the user's need, these parts can be easily selected and combined from a repository, serving as a toolkit for rapid genome editing without any expensive reagent. The toolkit contains verified linear DNA fragments, which are easy to store, share, and transport at room temperature, drastically reducing expensive shipping costs and assembly time. After optimizing this technique, eight loci proximal to autonomously replicating sequences (ARS) in the yeast genome were also characterized in terms of integration and gene expression efficiencies and the impacts of the disruptions of these regions on cell fitness. The flexibility and multiplexing capacity of the ACtivE were shown by constructing a β-carotene pathway. In only a few days, >80% integration efficiency for single gene integration and >50% integration efficiency for triplex integration were achieved on Saccharomyces cerevisiae BY4741 from scratch without using in vitro DNA assembly methods, restriction enzymes, or extra cloning steps. This study presents a standardizable method to be readily employed to accelerate yeast genome engineering and provides well-defined genomic location alternatives for yeast synthetic biology and metabolic engineering purposes.

Karyon: a computational framework for the diagnosis of hybrids, aneuploids, and other nonstandard architectures in genome assemblies.

BackgroundRecent technological developments have made genome sequencing and assembly highly accessible and widely used. However, the presence in sequenced organisms of certain genomic features such as high heterozygosity, polyploidy, aneuploidy, heterokaryosis, or extreme compositional biases can challenge current standard assembly procedures and result in highly fragmented assemblies. Hence, we hypothesized that genome databases must contain a nonnegligible fraction of low-quality assemblies that result from such type of intrinsic genomic factors.FindingsHere we present Karyon, a Python-based toolkit that uses raw sequencing data and de novo genome assembly to assess several parameters and generate informative plots to assist in the identification of nonchanonical genomic traits. Karyon includes automated de novo genome assembly and variant calling pipelines. We tested Karyon by diagnosing 35 highly fragmented publicly available assemblies from 19 different Mucorales (Fungi) species.ConclusionsOur results show that 10 (28.57%) of the assemblies presented signs of unusual genomic configurations, suggesting that these are common, at least for some lineages within the Fungi.

GBZ file format for pangenome graphs.

Pangenome graphs representing aligned genome assemblies are being shared in the text-based Graphical Fragment Assembly format. As the number of assemblies grows, there is a need for a file format that can store the highly repetitive data space efficiently. We propose the GBZ file format based on data structures used in the Giraffe short-read aligner. The format provides good compression, and the files can be efficiently loaded into in-memory data structures. We provide compression and decompression tools and libraries for using GBZ graphs, and we show that they can be efficiently used on a variety of systems. C++ and Rust implementations are available at https://github.com/jltsiren/gbwtgraph and https://github.com/jltsiren/gbwt-rs, respectively. Supplementary data are available at Bioinformatics online.

A chromosome-scale reference genome for Spironucleus salmonicida

Spironucleus salmonicida is a diplomonad causing systemic infection in salmon. The first S. salmonicida genome assembly was published 2014 and has been a valuable reference genome in protist research. However, the genome assembly is fragmented without assignment of the sequences to chromosomes. In our previous Giardia genome study, we have shown how a fragmented genome assembly can be improved with long-read sequencing technology complemented with optical maps. Combining Pacbio long-read sequencing technology and optical maps, we are presenting here this new S. salmonicida genome assembly in nine near-complete chromosomes with only three internal gaps at long repeats. This new genome assembly is not only more complete sequence-wise but also more complete at annotation level, providing more details into gene families, gene organizations and chromosomal structure. This near-complete reference genome will aid comparative genomics at chromosomal level, and serve as a valuable resource for the diplomonad community and protist research.

Fast and accurate Ab Initio Protein structure prediction using deep learning potentials.

Despite the immense progress recently witnessed in protein structure prediction, the modeling accuracy for proteins that lack sequence and/or structure homologs remains to be improved. We developed an open-source program, DeepFold, which integrates spatial restraints predicted by multi-task deep residual neural-networks along with a knowledge-based energy function to guide its gradient-descent folding simulations. The results on large-scale benchmark tests showed that DeepFold creates full-length models with accuracy significantly beyond classical folding approaches and other leading deep learning methods. Of particular interest is the modeling performance on the most difficult targets with very few homologous sequences, where DeepFold achieved an average TM-score that was 40.3% higher than trRosetta and 44.9% higher than DMPfold. Furthermore, the folding simulations for DeepFold were 262 times faster than traditional fragment assembly simulations. These results demonstrate the power of accurately predicted deep learning potentials to improve both the accuracy and speed of ab initio protein structure prediction.

FebRNA: An automated fragment-ensemble-based model for building RNA 3D structures

Knowledge of RNA three-dimensional (3D) structures is critical to understanding the important biological functions of RNAs. Although various structure prediction models have been developed, the high-accuracy predictions of RNA 3D structures are still limited to the RNAs with short lengths or with simple topology. In this work, we proposed a new model, namely FebRNA, for building RNA 3D structures through fragment assembly based on coarse-grained (CG) fragment ensembles. Specifically, FebRNA is composed of four processes: establishing the library of different types of non-redundant CG fragment ensembles regardless of the sequences, building CG 3D structure ensemble through fragment assembly, identifying top-scored CG structures through a specific CG scoring function, and rebuilding the all-atom structures from the top-scored CG ones. Extensive examination against different types of RNA structures indicates that FebRNA consistently gives the reliable predictions on RNA 3D structures, including pseudoknots, three-way junctions, four-way and five-way junctions, and RNAs in the RNA-Puzzles. FebRNA is available on the Web site: https://github.com/Tan-group/FebRNA.

Graph-based Automated Macro-Molecule Assembly.

We present a general molecular framework assembly algorithm that takes a largely arbitrary molecular fragment database and a user-supplied target template graph as input. Automatic assembly of molecular fragments from the database, following a prescribed, user-supplied set of connection rules, then turns the template graph into an actual, chemically reasonable molecular framework. Assembly capabilities of our algorithm are tested by producing several abstract, closed-loop shapes. To indicate a few of many possible application areas we demonstrate a host-guest complex and a road toward catalysis. Postassembly substituent exchange can be used to produce electric fields of desired values at desired points inside the framework or at its surface as a stepping stone toward rationally designed, artificial heterogeneous catalysts.

Sequencing of Enteric Bacteria: Library Preparation Procedure Matters for Accurate Identification and Characterization.

Enzymatic library preparation kits are increasingly used for bacterial whole genome sequencing. While they offer a rapid workflow, the transposases used in the kits are recognized to be somewhat biased. The aim of this study was to optimize and validate a protocol for the Illumina DNA Prep kit (formerly Nextera DNA Flex) for sequencing enteric pathogens and compare its performance against the Nextera XT kit. One hundred forty-three strains of Campylobacter, Escherichia, Listeria, Salmonella, Shigella, and Vibrio were prepared with both methods and sequenced on the Illumina MiSeq using 300 and/or 500 cycle chemistries. Sequences were compared using core genome multilocus sequence typing (cgMLST), 7-gene multilocus sequence typing (MLST), and detection of markers encoding serotype, virulence, and antimicrobial resistance. Sequences for one Escherichia strain were downsampled to determine the minimum coverage required for the analyses. While organism-specific differences were observed, the Prep libraries generated longer average read lengths and less fragmented assemblies compared to the XT libraries. In downstream analysis, the most notable difference between the kits was observed for Escherichia, particularly for the 300 cycle sequences. The O group was not predicted in 32% and 4% of XT sequences when using blast and kmer algorithms, respectively, while the O group was predicted from all Prep sequences regardless of the algorithm. In addition, the ehxA gene was not detected in 6% of XT sequences and 34% were missing one or more of the type III secretion systems and/or plasmid-associated genes, which were detected in the Prep sequences. The coverage downsampling revealed that acceptable assembly quality and allele detection was achieved at 30 × coverage with the Prep libraries, whereas 40-50 × coverage was required for the XT libraries. The better performance of the Prep libraries was attributed to more even coverage, particularly in genome regions low in GC content.

Selective and Reversible Ligand Assembly on the DNA and RNA Repeat Sequences in Myotonic Dystrophy.

Small molecule targeting of DNA and RNA sequences has come into focus as a therapeutic strategy for diseases such as myotonic dystrophy type 1 (DM1), a trinucleotide repeat disease characterized by RNA gain-of-function. Herein, we report a novel template-selected, reversible assembly of therapeutic agents in situ via aldehyde-amine condensation. Rationally designed small molecule targeting agents functionalized with either an aldehyde or an amine were synthesized and screened against the target nucleic acid sequence. The assembly of fragments was confirmed by MALDI-MS in the presence of DM1-relevant nucleic acid sequences. The resulting hit combinations of aldehyde and amine inhibited the formation of r(CUG)exp in vitro in a cooperative manner at low micromolar levels and rescued mis-splicing defects in DM1 model cells. This reversible template-selected assembly is a promising approach to achieve cell permeable and multivalent targeting via in situ synthesis and could be applied to other nucleic acid targets.

Synthesis of Pleuromutilin.

Synthesis of a potent inhibitor of bacterial protein synthesis, pleuromutilin, is described. Assembly of the critical cyclooctane fragment relies on an oxidative ring-expansion, and complete stereochemical relay in the synthetic sequence is enabled by the judicious choice of tactics. The requisite connectivity pattern of the perhydroindanone motif is rapidly established in a sequence of cycloaddition and radical cyclization events. Application of this strategy allows for preparation of the target natural product in 16 steps from commercially available material.

Genome of the lepidopleurid chiton Hanleya hanleyi (Mollusca, Polyplacophora).

Mollusca is the second most species-rich phylum and includes animals as disparate as octopuses, clams, and chitons. Dozens of molluscan genomes are available, but only one representative of the subphylum Aculifera, the sister taxon to all other molluscs, has been sequenced to date, hindering comparative and evolutionary studies. To facilitate evolutionary studies across Mollusca, we sequenced the genome of a second aculiferan mollusc, the lepidopleurid chiton Hanleya hanleyi (Bean 1844), using a hybrid approach combining Oxford Nanopore and Illumina reads. After purging redundant haplotigs and removing contamination from this 1.3% heterozygous genome, we produced a 2.5 Gbp haploid assembly (>4X the size of the other chiton genome sequenced to date) with an N50 of 65.0 Kbp. Despite a fragmented assembly, the genome is rather complete (92.0% of BUSCOs detected; 79.4% complete plus 12.6% fragmented). Remarkably, the genome has the highest repeat content of any molluscan genome reported to date (>66%). Our gene annotation pipeline predicted 69,284 gene models (92.9% of BUSCOs detected; 81.8% complete plus 11.1% fragmented) of which 35,362 were supported by transcriptome and/or protein evidence. Phylogenomic analysis recovered Polyplacophora sister to all other sampled molluscs with maximal support. The Hanleya genome will be a valuable resource for studies of molluscan biology with diverse potential applications ranging from evolutionary and comparative genomics to molecular ecology.

Novel Multiplex PCR Method and Genome Sequence-Based Analog for High-Resolution Subclonal Assignment and Characterization of Escherichia coli Sequence Type 131 Isolates.

ABSTRACTEscherichia coli sequence type 131 (ST131) is a pandemic, multidrug-resistant extraintestinal pathogen. The multiple distinctive ST131 subclones differ for rfb and fliC alleles (O and H antigens), fimH allele (type-1 fimbriae adhesin), resistance phenotype and genotype, clinical correlates, and host predilection. Current PCR assays for detecting ST131 and its main subclones offer limited sub-ST characterization. Here we combined 22 novel and 14 published primers for a multiplex PCR assay to detect and extensively characterize ST131 isolates. The primers target mdh36, gyrB47, trpA72, sbmA, plsB, nupC, rmuC, kefC, ybbW, the O16 and O25b rfb variants, five fimH alleles (fimH22, fimH27, fimH30, fimH35, and fimH41), two fliC alleles (H4 and H5), a (subclone-specific) fluoroquinolone resistance-associated parC allele, and a (subclone-specific) prophage marker. The resulting amplicons resolve 15 molecular subsets within ST131, including 3 within clade A (H41 subclone), 5 within clade B (H22 subclone), and 7 within clade C (H30 subclone), which includes subclones C0 (H30S: 2 subsets), C1 and C1-M27 (H30R1: 2 subsets), and C2 (H30Rx: 3 subsets). Validation in three laboratories showed that this assay provides a rapid, accurate, and portable method for rapidly detecting and characterizing E. coli ST131 and its key subsets. Additionally, for users with whole genome sequencing (WGS) capability, we developed a command-line executable called ST131Typer, an in silico version of the extended multiplex PCR assay. Its accuracy was 87.8%, with most issues due to incomplete or fragmented input genome assemblies. These two novel assays should facilitate detailed ST131 subtyping using either endpoint PCR or WGS.IMPORTANCE These novel assays provide greater subclonal resolution and characterization of E. coli ST131 isolates than do the available comparable PCR assays, plus offer a novel sequence-based alternative to PCR. They may prove useful for molecular epidemiological studies, surveillance, and, potentially, clinical management.

ODGI: understanding pangenome graphs.

MotivationPangenome graphs provide a complete representation of the mutual alignment of collections of genomes. These models offer the opportunity to study the entire genomic diversity of a population, including structurally complex regions. Nevertheless, analyzing hundreds of gigabase-scale genomes using pangenome graphs is difficult as it is not well-supported by existing tools. Hence, fast and versatile software is required to ask advanced questions to such data in an efficient way.ResultsWe wrote Optimized Dynamic Genome/Graph Implementation (ODGI), a novel suite of tools that implements scalable algorithms and has an efficient in-memory representation of DNA pangenome graphs in the form of variation graphs. ODGI supports pre-built graphs in the Graphical Fragment Assembly format. ODGI includes tools for detecting complex regions, extracting pangenomic loci, removing artifacts, exploratory analysis, manipulation, validation and visualization. Its fast parallel execution facilitates routine pangenomic tasks, as well as pipelines that can quickly answer complex biological questions of gigabase-scale pangenome graphs.Availability and implementationODGI is published as free software under the MIT open source license. Source code can be downloaded from https://github.com/pangenome/odgi and documentation is available at https://odgi.readthedocs.io. ODGI can be installed via Bioconda https://bioconda.github.io/recipes/odgi/README.html or GNU Guix https://github.com/pangenome/odgi/blob/master/guix.scm.Supplementary information Supplementary data are available at Bioinformatics online.

A split green fluorescent protein system to enhance spatial and temporal sensitivity of translating ribosome affinity purification.

SUMMARYTranslating ribosome affinity purification (TRAP) utilizes transgenic plants expressing a ribosomal protein fused to a tag for affinity co‐purification of ribosomes and the mRNAs that they are translating. This population of actively translated mRNAs (translatome) can be interrogated by quantitative PCR or RNA sequencing. Condition‐ or cell‐specific promoters can be utilized to isolate the translatome of specific cell types, at different growth stages and/or in response to environmental variables. While advantageous for revealing differential expression, this approach may not provide sufficient sensitivity when activity of the condition/cell‐specific promoter is weak, when ribosome turnover is low in the cells of interest, or when the targeted cells are ephemeral. In these situations, expressing tagged ribosomes under the control of these specific promoters may not yield sufficient polysomes for downstream analysis. Here, we describe a new TRAP system that employs two transgenes: One is constitutively expressed and encodes a ribosomal protein fused to one fragment of a split green fluorescent protein (GFP); the second is controlled by a stimulus‐specific promoter and encodes the second GFP fragment fused to an affinity purification tag. In cells where both transgenes are active, the purification tag is attached to ribosomes by bi‐molecular folding and assembly of the split GFP fragments. This approach provides increased sensitivity and better temporal resolution because it labels pre‐existing ribosomes and does not depend on rapid ribosome turnover. We describe the optimization and key parameters of this system, and then apply it to a plant–pathogen interaction in which spatial and temporal resolution are difficult to achieve with current technologies.

PhyloHerb: A high-throughput phylogenomic pipeline for processing genome skimming data.

PremiseThe application of high‐throughput sequencing, especially to herbarium specimens, is rapidly accelerating biodiversity research. Low‐coverage sequencing of total genomic DNA (genome skimming) is particularly promising and can simultaneously recover the plastid, mitochondrial, and nuclear ribosomal regions across hundreds of species. Here, we introduce PhyloHerb, a bioinformatic pipeline to efficiently assemble phylogenomic data sets derived from genome skimming.Methods and ResultsPhyloHerb uses either a built‐in database or user‐specified references to extract orthologous sequences from all three genomes using a BLAST search. It outputs FASTA files and offers a suite of utility functions to assist with alignment, partitioning, concatenation, and phylogeny inference. The program is freely available at https://github.com/lmcai/PhyloHerb/.ConclusionsWe demonstrate that PhyloHerb can accurately identify genes using a published data set from Clusiaceae. We also show via simulations that our approach is effective for highly fragmented assemblies from herbarium specimens and is scalable to thousands of species.