Assembling DNA Fragments Research Articles

Validation and Application of Long-Read Whole-Genome Sequencing for Antimicrobial Resistance Gene Detection and Antimicrobial Susceptibility Testing.

Next-generation sequencing applications are increasingly used for detection and characterization of antimicrobial-resistant pathogens in clinical settings. Oxford Nanopore Technologies (ONT) sequencing offers advantages for clinical use compared with other sequencing methodologies because it enables real-time basecalling, produces long sequencing reads that increase the ability to correctly assemble DNA fragments, provides short turnaround times, and requires relatively uncomplicated sample preparation. A drawback of ONT sequencing, however, is its lower per-read accuracy than short-read sequencing. We sought to identify best practices in ONT sequencing protocols. As some variability in sequencing results may be introduced by the DNA extraction methodology, we tested three DNA extraction kits across three independent laboratories using a representative set of six bacterial isolates to investigate accuracy and reproducibility of ONT technology. All DNA extraction techniques showed comparable performance; however, the DNeasy PowerSoil Pro kit had the highest sequencing yield. This kit was subsequently applied to 42 sequentially collected bacterial isolates from blood cultures to assess Ares Genetics's pipelines for predictive whole-genome sequencing antimicrobial susceptibility testing (WGS-AST) performance compared to phenotypic triplicate broth microdilution results. WGS-AST results ranged across the organisms and resulted in an overall categorical agreement of 95% for penicillins, 82.4% for cephalosporins, 76.7% for carbapenems, 86.9% for fluoroquinolones, and 96.2% for aminoglycosides. Very major errors/major errors were 0%/16.7% (penicillins), 11.7%/3.6% (cephalosporins), 0%/24.4% (carbapenems), 2.5%/7.7% (fluoroquinolones), and 0%/4.1% (aminoglycosides), respectively. This work showed that, although additional refinements are necessary, ONT sequencing demonstrates potential as a method to perform WGS-AST on cultured isolates for patient care.

Impact of Synonymous Genome Recoding on the HIV Life Cycle.

Synonymous mutations within protein coding regions introduce changes in DNA or messenger (m) RNA, without mutating the encoded proteins. Synonymous recoding of virus genomes has facilitated the identification of previously unknown virus biological features. Moreover, large-scale synonymous recoding of the genome of human immunodeficiency virus type 1 (HIV-1) has elucidated new antiviral mechanisms within the innate immune response, and has improved our knowledge of new functional virus genome structures, the relevance of codon usage for the temporal regulation of viral gene expression, and HIV-1 mutational robustness and adaptability. Continuous improvements in our understanding of the impacts of synonymous substitutions on virus phenotype – coupled with the decreased cost of chemically synthesizing DNA and improved methods for assembling DNA fragments – have enhanced our ability to identify potential HIV-1 and host factors and other aspects involved in the infection process. In this review, we address how silent mutagenesis impacts HIV-1 phenotype and replication capacity. We also discuss the general potential of synonymous recoding of the HIV-1 genome to elucidate unknown aspects of the virus life cycle, and to identify new therapeutic targets.

Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research.

Plant genetic engineering vectors, such as RNA interference (RNAi) and CRISPR/Cas9 vectors, are important tools for plant functional genomics. Efficient construction of these functional vectors can facilitate the study of gene function. Although some methods for vector construction have been reported, their operations are still complicated and costly. Here, we describe a simpler and low-cost vector construction method by nicking endonucleases-mediated DNA assembly (NEMDA), which uses nicking endonucleases to generate single-strand overhanging complementary ends for rapid assembly of DNA fragments into plasmids. Using this approach, we rapidly completed the construction of four RNAi vectors and a CRISPR/Cas9 knockout vector with five single-guide RNA (sgRNA)-expression cassettes for multiplex genome editing, and successfully achieved the goal of decreasing the expression of the target genes and knocking out the target genes at the same time in rice. These results indicate the great potential of NEMDA in assembling DNA fragments and constructing plasmids for molecular biology and functional genomics.

An efficient method for the construction of artificial, concatemeric DNA, RNA and proteins with genetically programmed functions, using a novel, vector-enzymatic DNA fragment amplification-expression technology

De novo designed bioactive molecules, such as DNA, RNA and peptides, are utilized in increasingly diverse scientific, industrial and biomedical applications. Concatemerization of designed DNA, RNA and peptides may improve their stability, bioactivity and allow for gradual release of the bioactive molecule at the intended destination. In this context, we developed a new method enabling the formation of DNA concatemers for the production of artificial, repetitive genes, encoding concatemeric RNAs and proteins of any nucleotide and amino-acid sequence. The technology recruits the Type IIS SapI restriction endonuclease (REase) for assembling DNA fragments in an ordered head-to-tail-orientation. Alternatively, other commercially available SapI isoschizomers can be used: LguI and thermostable BspQI. Four series of DNA vectors dedicated to the expression of newly formed, concatemeric open reading frames (ORFs), were designed and constructed to meet the technology needs.• Vector-enzymatic DNA fragment amplification technology.• Construction of DNA concatemers many times longer than those available with the use of current de novo gene synthesis methods.• Biosynthesis of protein tandem repeats with programmable function never seen in nature.

Hierarchical Overlap Graph

Given a set of finite words, the Overlap Graph (OG) is a complete weighted digraph where each word is a node and where the weight of an arc equals the length of the longest overlap of one word onto the other (Overlap is an asymmetric notion). The OG serves to assemble DNA fragments or to compute shortest superstrings, which are a compressed representation of the input. The OG requires space that is quadratic in the number of words, which limits its scalability. The Hierarchical Overlap Graph (HOG) is an alternative graph that also encodes all maximal overlaps, but uses space that is linear in the sum of the lengths of the input words. We propose the first algorithm to build the HOG in linear space for words of equal length.

Synonymous genome recoding: a tool to explore microbial biology and new therapeutic strategies.

Synthetic genome recoding is a new means of generating designed organisms with altered phenotypes. Synonymous mutations introduced into the protein coding region tolerate modifications in DNA or mRNA without modifying the encoded proteins. Synonymous genome-wide recoding has allowed the synthetic generation of different small-genome viruses with modified phenotypes and biological properties. Recently, a decreased cost of chemically synthesizing DNA and improved methods for assembling DNA fragments (e.g. lambda red recombination and CRISPR-based editing) have enabled the construction of an Escherichia coli variant with a 4-Mb synthetic synonymously recoded genome with a reduced number of sense codons (n = 59) encoding the 20 canonical amino acids. Synonymous genome recoding is increasing our knowledge of microbial interactions with innate immune responses, identifying functional genome structures, and strategically ameliorating cis-inhibitory signaling sequences related to splicing, replication (in eukaryotes), and complex microbe functions, unraveling the relevance of codon usage for the temporal regulation of gene expression and the microbe mutant spectrum and adaptability. New biotechnological and therapeutic applications of this methodology can easily be envisaged. In this review, we discuss how synonymous genome recoding may impact our knowledge of microbial biology and the development of new and better therapeutic methodologies.

Machine learning meets genome assembly.

With the recent advances in DNA sequencing technologies, the study of the genetic composition of living organisms has become more accessible for researchers. Several advances have been achieved because of it, especially in the health sciences. However, many challenges which emerge from the complexity of sequencing projects remain unsolved. Among them is the task of assembling DNA fragments from previously unsequenced organisms, which is classified as an NP-hard (nondeterministic polynomial time hard) problem, for which no efficient computational solution with reasonable execution time exists. However, several tools that produce approximate solutions have been used with results that have facilitated scientific discoveries, although there is ample room for improvement. As with other NP-hard problems, machine learning algorithms have been one of the approaches used in recent years in an attempt to find better solutions to the DNA fragment assembly problem, although still at a low scale. This paper presents a broad review of pioneering literature comprising artificial intelligence-based DNA assemblers-particularly the ones that use machine learning-to provide an overview of state-of-the-art approaches and to serve as a starting point for further study in this field.

Identification and heterologous expression of the actinoallolide biosynthetic gene cluster.

Actinoallolides are anti-trypanosomal macrolides isolated from the secondary metabolites of two endophytic actinomycete strains, Actinoallomurus fulvus MK10-036 and K09-0307. A putative actinoallolide biosynthetic gene cluster was predicted from the genome sequence of the strain K09-0307. The gene cluster spans a contiguous 53 kb DNA region that comprises seven genes encoding three PKSs (aalA1, aalA2, and aalA3), cytochrome P450 (aalB), acyl-CoA dehydrogenase (aalC), crotonyl-CoA reductase (aalD), and TetR family regulator (aalR). The entire gene cluster was cloned into a plasmid pYIK1 by assembling DNA fragments, which were obtained from two cosmids containing left and right parts of the gene cluster. Following the introduction of an ermE* promoter at 100bp upstream from the start codon of aalA1, the gene cluster was introduced into Streptomyces coelicolor M1152. Subsequent LC-MS analysis revealed production of actinoallolide A in the culture broth. Thus, the actinoallolide biosynthetic gene cluster was identified by heterologous expression in Streptomyces.

A method to assemble DNA fragments mimicking junctions of transgenic elements: Application to the AquAdvantage salmon

The detection of transgenic animals in food and environmental samples will require the development of reliable screening assays. The identification of junction regions between genetic elements is the unequivocal way to prove the presence of transgene constructs. However, a sample from the transgenic organism or genetic construct is not always available for analysis. Here, we describe an easy-to-implement method to create junctions of genetic elements that can serve as DNA template or positive control for the development and validation of detection methods. The new methodology was successfully used to test several pairs of PCR primers intended for the detection of the first transgenic animal approved for human consumption (the AquAdvantage salmon). The junction region was created by the PCR amplification of a genetic element (antifreeze protein gene) with a tailed primer containing part of the adjacent element (growth hormone gene), yielding a PCR product with both regions fused. This strategy can be adapted to create junctions of genetic elements in any transgenic organism if reliable sequence data is available.

Sequence-independent cloning methods for long DNA fragments applied to synthetic biology

Simplified methods to assemble DNA fragments by independent cloning sequence have helped in the progress of synthetic biology, allowing some biotechnological processes to become economically viable by genetic improvement of microorganisms. We compared three methods of assembling six DNA fragments: PCR fusion-based, isothermal NEBuilder and circular polymerase extension cloning (CPEC). Double and triple fusion occurs directly with the PCR products using PCR fusion-based and NEBuilder methods. For multiple fragments the results showed higher efficiency by the CPEC method which allowed assembly of six fragments previously purified by agarose gel extraction, after a sequence of 20 annealing/extension cycles without any primer.

An accurate DNA sequence assembly algorithm based on MapReduce

DNA sequence assembly and gene homology comparison are the most basic and most frequently used methods in bioinformatics, which can be used to estimate the genetic integrity, gene structure and function. These years, a lot of scholars have done a lot of work in the direction of sequence assembly al gorithm, and put forward some effective methods. However, accurate and efficient assembly algorithm is very rare. In order to achieve accuracy and efficiency, this paper proposes an accurate and efficient algorithm based on MapReduce. The algorithm makes full use of the advantages of the map and reduce functions, and the experimental results show that it can quickly and accurately assemble DNA fragments no matter small scale sequences or middle-scale genome sequences.

A simple and ultra-low cost homemade seamless ligation cloning extract (SLiCE) as an alternative to a commercially available seamless DNA cloning kit

The seamless ligation cloning extract (SLiCE) method is a novel seamless DNA cloning tool that utilizes homologous recombination activities in Escherichia coli cell lysates to assemble DNA fragments into a vector. Several laboratory E. coli strains can be used as a source for the SLiCE extract; therefore, the SLiCE-method is highly cost-effective.The SLiCE has sufficient cloning ability to support conventional DNA cloning, and can simultaneously incorporate two unpurified DNA fragments into vector. Recently, many seamless DNA cloning kits have become commercially available; these are generally very convenient, but expensive. In this study, we evaluated the cloning efficiencies between a simple and highly cost-effective SLiCE-method and a commercial kit under various molar ratios of insert DNA fragments to vector DNA. This assessment identified that the SLiCE from a laboratory E. coli strain yielded 30−85% of the colony formation rate of a commercially available seamless DNA cloning kit. The cloning efficiencies of both methods were highly effective, exhibiting over 80% success rate under all conditions examined. These results suggest that SLiCE from a laboratory E. coli strain can efficiently function as an effective alternative to commercially available seamless DNA cloning kits.

A Versatile Microfluidic Device for Automating Synthetic Biology.

New microbes are being engineered that contain the genetic circuitry, metabolic pathways, and other cellular functions required for a wide range of applications such as producing biofuels, biobased chemicals, and pharmaceuticals. Although currently available tools are useful in improving the synthetic biology process, further improvements in physical automation would help to lower the barrier of entry into this field. We present an innovative microfluidic platform for assembling DNA fragments with 10× lower volumes (compared to that of current microfluidic platforms) and with integrated region-specific temperature control and on-chip transformation. Integration of these steps minimizes the loss of reagents and products compared to that with conventional methods, which require multiple pipetting steps. For assembling DNA fragments, we implemented three commonly used DNA assembly protocols on our microfluidic device: Golden Gate assembly, Gibson assembly, and yeast assembly (i.e., TAR cloning, DNA Assembler). We demonstrate the utility of these methods by assembling two combinatorial libraries of 16 plasmids each. Each DNA plasmid is transformed into Escherichia coli or Saccharomyces cerevisiae using on-chip electroporation and further sequenced to verify the assembly. We anticipate that this platform will enable new research that can integrate this automated microfluidic platform to generate large combinatorial libraries of plasmids and will help to expedite the overall synthetic biology process.

Increase in the astaxanthin synthase gene (crtS) dose by in vivo DNA fragment assembly in Xanthophyllomyces dendrorhous.

BackgroundXanthophyllomyces dendrorhous is a basidiomycetous yeast that is relevant to biotechnology, as it can synthesize the carotenoid astaxanthin. However, the astaxanthin levels produced by wild-type strains are low. Although different approaches for promoting increased astaxanthin production have been attempted, no commercially competitive results have been obtained thus far. A promising alternative to facilitate the production of carotenoids in this yeast involves the use of genetic modification. However, a major limitation is the few available molecular tools to manipulate X. dendrorhous.ResultsIn this work, the DNA assembler methodology that was previously described in Saccharomyces cerevisiae was successfully applied to assemble DNA fragments in vivo and integrate these fragments into the genome of X. dendrorhous by homologous recombination in only one transformation event. Using this method, the gene encoding astaxanthin synthase (crtS) was overexpressed in X. dendrorhous and a higher level of astaxanthin was produced.ConclusionsThis methodology could be used to easily and rapidly overexpress individual genes or combinations of genes simultaneously in X. dendrorhous, eliminating numerous steps involved in conventional cloning methods.

Building bugs

Building bugs

Sticky egyptians: a technique for assembling genes encoding constrained peptides of variable length.

Naturally occurring peptides, such as those produced by the poisonous marine snails of the genus Conus , have the ability to form tight, highly specific molecular interactions. The rigidity of the peptide framework which promotes these interactions is usually maintained by disulphide bonds, and it seems that the overall main chain conformation (or fold) of the peptide is determined by its length and the sequence distribution of the pairs of cysteine residues participating in these bonds. The fold of the peptide in turn is largely responsible for its shape. Since highly effective molecular interactions occur between species complementary in shape, we reasoned that peptides with the greatest potential in therapy or diagnosis would be found in a library of shapes, those peptides with a shape complementary to a given target being identified, for example, by selection. As a first step towards constructing such a peptide shape library, we have developed a method for assembling DNA fragments which encode an even number of cysteine residues and which are of variable length. We describe this method here.

A contig assembly program based on sensitive detection of fragment overlaps

An effective computer program for assembling DNA fragments, the contig assembly program (CAP), has been developed. In the CAP program, a filter is used to eliminate quickly fragment pairs that could not possibly overlap, a dynamic programming algorithm is applied to compute the maximal-scoring overlapping alignment between each remaining pair of fragments, and a simple greedy approach is employed to assemble fragments in order of alignment scores. To identify the true fragment overlaps, the dynamic programming algorithm uses specially chosen sets of alignment parameters to tolerate sequencing errors and to penalize "mutational" changes between different copies of a repetitive sequence. The performance tests of the program on fragment data from genomic sequencing projects produced satisfactory results. The CAP program is efficient in computer time and memory; it took about 4 h to assemble a set of 1015 fragments into long contigs on a Sun workstation.

ChemInform Abstract: SYNTHESIS OF DNA FRAGMENTS CONTAINING 5‐METHYL‐DEOXYCYTIDINE RESIDUES

AbstractAusgehend ‐vom Thymidin‐Deiivat (I) wird über die Stufen (IIa)‐(IId) das phosphorylierte S‐Methyl‐ 2′‐desoxy‐cytidin (IIe) dargestellt, das weiter in das Derivat (IIf) sowie dessen Deblokkierungsprodukt (III) übergeführt wird.

Synthesis of DNA fragments containing 5‐methyl‐deoxycytidine residues

AbstractA properly protected 5‐methylated deoxycytidine (5mC) derivative has been synthesized and applied to assemble DNA fragments containing 5mCG, 5mCC and 5mC5mC sequences.