Combination Of Nanopore Research Articles

Chromosome-Level Assembly Reveals a Fifteen-Chromosome Aneuploid Genome and Environmental Adaptation Strategy of Chinese Traditional Medical Fungus Wolfiporia hoelen.

The sclerotia of Wolfiporia hoelen are one of the most important traditional Chinese medicines and foods commonly used in China, Japan, Korea, and other Asian countries. To provide a high-quality reference genome and deepen our understanding of the genome of W. hoelen to elucidate various biological phenomena. In this study, we assembled three genomes of W. hoelen using a combination of Nanopore and Illumina sequencing strategies. The fifteen-chromosome genome L7 of W. hoelen was assembled with two-sided telomere and rDNA sequences for the first time. The chromosome count was subsequently confirmed through collinearity analysis, correcting the previous belief that W. hoelen had only fourteen chromosomes. Moreover, the aneuploid genome was discovered in W. hoelen for the first time through sequencing depth analysis of different chromosomes, and only some strains of W. hoelen exhibit aneuploid genomes. According to the genome analysis of homokaryotic offspring and protoplast-isolated strains, a potential variation in chromosome allocation patterns was revealed. Moreover, the gene function enrichment analysis of genes on reduplicated chromosomes demonstrated that aneuploidy in the genome may be the result of environmental adaptation for W. hoelen. The discovery of an aneuploid genome also provides new ideas for genetic improvement of W. hoelen.

The complete genome of Escherichia coli JM101 assembled with a combination of Nanopore and Illumina platforms.

We report the complete genome and the plasmid (F' episome) sequences of Escherichia coli JM101 assembled with a combination of Nanopore and Illumina data. The resulting genome is a single contig of 4,524,963 bp, and the plasmid consists of a single contig of 197,186 bp.

Nanopores limited domain and PVA film used to assist the SERS property of gold nanoparticle arrays

The combination of nanopores and noble metal nanoparticles has been the focus of research in recent years, and their synergistic effect enabled the substrate exhibit excellent SERS property. In this paper, the array porous silicon structures embedded with gold nanoparticles have been prepared by using the polystyrene (PS) microsphere template assisted gold annealing process and metal assisted chemical etching techniques (AuNPs@nanopore). Polyvinyl alcohol (PVA) thin film was introduced as a molecular adsorption layer to be spin coated on the surface and formed composite structure (AuNPs@nanopore @PVA) to enrich more detection molecules. Through the SERS analysis of different depths of gold nanoparticles by Rhodamine 6G (R6G) probe molecules, the optimal etching time, that is, the optimal depth that gold nanoparticles should be in, was determined. Under the etching time of 2 min, the AuNPs@nanopore @PVA structure base with pore depth of 245 nm and diameter of gold nanoparticles of 181 nm showed high sensitivity, high uniformity and the best SERS property. It can realize the low concentration detection of 10−10 mol/L R6G molecule and 10−11 mol/L methylene blue (MB) molecule, and the enhancement factor is up to 1.20 × 107. The substrate can also be used to detect the pesticide thiabendazole molecule, the detection limit of 10−8 mol/L.

Trichocoleus desertorum isolated from Negev desert petroglyphs: Characterization, adaptation and bioerosion potential

The Negev petroglyphs are considered valuable cultural heritage sites, but unfortunately, they are exposed to deterioration processes caused by anthropogenic and natural forces. Despite the many studies that have already pointed to the role of cyanobacteria in biogenic rock weathering, the knowledge involved in the process is still lacking. In this study, a cyanobacterial strain was isolated from the Negev Desert petroglyphs aiming to reveal its involvement in geochemical cycles and in the weathering processes of the rock substrate. The strain was characterized using morphological, molecular, and microscopic studies. The morphological research revealed a green-bluish, bundle-forming filamentous strain characterized by trichomes embedded in a common sheath. A combination of Nanopore and Illumina sequencing technologies facilitated the assembly of a near-complete genome containing 5,458,034 base pairs. A total of 5027 coding sequences were revealed by implementing PROKKA software. Annotation of five replicas of the 16S ribosomal RNA genes revealed that the Negev cyanobacteria isolate is closely (99.73 %) related to Trichocoleus desertorum LSB90_MW403957 isolated from the Sahara Desert, Algeria. The local strain was thus named Trichocoleus desertorum NBK24 CP116619. Several gene sequences that code for possible environmental adaptation mechanisms were found. Our study also identified genes for membrane transporters involved in the exchange of chemical elements, suggesting a possible interaction with rock minerals. Microscopic observations of T. desertorum NBK24 CP116619 infected onto calcareous stone slabs under laboratory conditions demonstrated the effect of the isolated cyanobacteria on stone surface degradation. In conclusion, the findings of this study further our understanding of terrestrial cyanobacterial genomes and functions and highlight the role of T. desertorum NBK24 CP116619 in stone weathering processes. This information may contribute to the creation of efficient restoration strategies for stone monuments affected by cyanobacteria.

A Reference Genome Assembly for the Spotted Flycatcher (Muscicapa striata).

The spotted flycatcher (Muscicapa striata) forms with the Mediterranean flycatcher (Muscicapa tyrrhenica) a newly recognized species pair of trans-Saharan migratory passerines. These flycatchers present a nested peripatric distribution, a pattern especially unusual among high dispersal species that questions the eco-evolutionary factors involved during the speciation process. Here, we present a genome assembly for M. striata assembled using a combination of Nanopore and Illumina sequences. The final assembly is 1.08 Gb long and consists of 4,779 contigs with an N50 of 3.2 Mb. The completeness of our M. striata genome assembly is supported by the number of BUSCO (95%) and ultraconserved element (UCE) (4889/5041; 97.0%) loci retrieved. This assembly showed high synteny with the Ficedula albicollis reference genome, the closest species for which a chromosome-scale reference genome is available. Several inversions were identified and will need to be investigated at the family level.

A chromosome-level genome assembly of an early matured aromatic Japonica rice variety Qigeng10 to accelerate rice breeding for high grain quality in Northeast China.

Early-matured aromatic japonica rice from the Northeast is the most popular rice commodity in the Chinese market. The Qigeng10 (QG10) was one of the varieties with the largest planting area in this region in recent years. It was an early-matured japonica rice variety with a lot of superior traits such as semi-dwarf, lodging resistance, long grain, aromatic and good quality. Therefore, a high-quality assembly of Qigeng10 genome is critical and useful for japonica research and breeding. In this study, we produced a high-precision QG10 chromosome-level genome by using a combination of Nanopore and Hi-C platforms. Finally, we assembled the QG10 genome into 77 contigs with an N50 length of 11.80 Mb in 27 scaffolds with an N50 length of 30.55 Mb. The assembled genome size was 378.31Mb with 65 contigs and constituted approximately 99.59% of the 12 chromosomes. We identified a total of 1,080,819 SNPs and 682,392 InDels between QG10 and Nipponbare. We also annotated 57,599 genes by the Ab initio method, homology-based technique, and RNA-seq. Based on the assembled genome sequence, we detected the sequence variation in a total of 63 cloned genes involved in grain yield, grain size, disease tolerance, lodging resistance, fragrance, and many other important traits. Finally, we identified five elite alleles (qTGW2Nipponbare , qTGW3Nanyangzhan , GW5IR24 , GW6Suyunuo , and qGW8Basmati385 ) controlling long grain size, four elite alleles (COLD1Nipponbare , bZIP73Nipponbare , CTB4aKunmingxiaobaigu , and CTB2Kunmingxiaobaigu ) controlling cold tolerance, three non-functional alleles (DTH7Kitaake , Ghd7Hejiang19 , and Hd1Longgeng31 ) for early heading, two resistant alleles (PiaAkihikari and Pid4Digu ) for rice blast, a resistant allele STV11Kasalath for rice stripe virus, an NRT1.1BIR24 allele for higher nitrate absorption activity, an elite allele SCM3Chugoku117 for stronger culms, and the typical aromatic gene badh2-E2 for fragrance in QG10. These results not only help us to better elucidate the genetic mechanisms underlying excellent agronomic traits in QG10 but also have wide-ranging implications for genomics-assisted breeding in early-matured fragrant japonica rice.

A CRISPR-based chromosomal-separation technique for Escherichia coli

BackgroundNatural life systems can be significantly modified at the genomic scale by human intervention, demonstrating the great innovation capacity of genome engineering. Large epi-chromosomal DNA structures were established in Escherichia coli cells, but some of these methods were inconvenient, using heterologous systems, or relied on engineered E. coli strains.ResultsThe wild-type model bacterium E. coli has a single circular chromosome. In this work, a novel method was developed to split the original chromosome of wild-type E. coli. With this method, novel E. coli strains containing two chromosomes of 0.10 Mb and 4.54 Mb, and 2.28 Mb and 2.36 Mb were created respectively, designated as E. coli0.10/4.54 and E. coli2.28/2.36. The new chromosomal arrangement was proved by PCR amplification of joint regions as well as a combination of Nanopore and Illumina sequencing analysis. While E. coli0.10/4.54 was quite stable, the two chromosomes of E. coli2.28/2.36 population recombined into a new chromosome (Chr.4.64MMut), via recombination. Both engineered strains grew slightly slower than the wild-type, and their cell shapes were obviously elongated.ConclusionFinally, we successfully developed a simple CRISPR-based genome engineering technique for the construction of multi-chromosomal E. coli strains with no heterologous genetic parts. This technique might be applied to other prokaryotes for synthetic biology studies and applications in the future.

The first high-quality chromosome-level genome assembly of Phyllanthaceae (Phyllanthus cochinchinensis) provides insights into flavonoid biosynthesis.

We report the genome assembly of P. cochinchinensis, as the first high-quality chromosome-level genome of Phyllanthaceae which is rich in medicinal plants. Phyllanthus cochinchinensis, a member of the Phyllanthaceae, is one of the famous medicinal plants in South China. Here, we report a de novo chromosome-level genome assembly for P. cochinchinensis using a combination of Nanopore and Illumina sequencing technologies. In total, the assembled genome consists of 284.88Mb genomic sequences with a contig N50 of 10.32Mb, representing ~ 95.49% of the estimated genome size. By applying Hi-C data, 13 pseudochromosomes of P. cochinchinensis were constructed, covering ~ 99.87% of the assembled sequences. The genome is annotated with 59.12% repetitive sequences and 20,836 protein-coding genes. Whole-genome duplication of P. cochinchinensis is likely shared with Ricinus communis as well as Vitis vinifera. Homologous genes within the flavonoid pathway for P. cochinchinensis were identified and copy numbers and expression level of related genes revealed potential critical genes involved in flavonoid biosynthesis. This study provides the first whole-genome sequence for the Phyllanthaceae, confirms the evolutionary status of Phyllanthus from the genomic level, and provides foundations for accelerating functional genomic research of species from Phyllanthus.

De novo genome assembly of the medicinal plant Gentiana macrophylla provides insights into the genomic evolution and biosynthesis of iridoids.

Gentiana macrophylla is a perennial herb in the Gentianaceae family, whose dried roots are used in traditional Chinese medicine. Here, we assembled a chromosome-level genome of G. macrophylla using a combination of Nanopore, Illumina, and Hi-C scaffolding approaches. The final genome size was ~1.79 Gb (contig N50 = 720.804 kb), and 98.89% of the genome sequences were anchored on 13 pseudochromosomes (scaffold N50 = 122.73 Mb). The genome contained 55,337 protein-coding genes, and 73.47% of the assemblies were repetitive sequences. Genome evolution analysis indicated that G. macrophylla underwent two rounds of whole-genome duplication after the core eudicot γ genome triplication event. We further identified candidate genes related to the biosynthesis of iridoids, and the corresponding gene families mostly expanded in G. macrophylla. In addition, we found that root-specific genes are enriched in pathways involved in defense responses, which may greatly improve the biological adaptability of G. macrophylla. Phylogenomic analyses showed a sister relationship of asterids and rosids, and all Gentianales species formed a monophyletic group. Our study contributes to the understanding of genome evolution and active component biosynthesis in G. macrophylla and provides important genomic resource for the genetic improvement and breeding of G. macrophylla.

The characterization of multiple novel paramyxoviruses highlights the diverse nature of the subfamily Orthoparamyxovirinae.

The subfamily Orthoparamyxovirinae is a group of single-stranded, negative-sense RNA viruses that contains many human, animal, and zoonotic pathogens. While there are currently only forty-two recognized species in this subfamily, recent research has revealed that much of its diversity remains to be characterized. Using a newly developed nested PCR-based screening assay, we report here the discovery of fifteen orthoparamyxoviruses in rodents and shrews from Belgium and Guinea, thirteen of which are believed to represent new species. Using a combination of nanopore and sanger sequencing, complete genomes could be determined for almost all these viruses, enabling a detailed evaluation of their genome characteristics. While most viruses are thought to belong to the rapidly expanding genus Jeilongvirus, we also identify novel members of the genera Narmovirus, Henipavirus, and Morbillivirus. Together with other recently discovered orthoparamyxoviruses, both henipaviruses and the morbillivirus discovered here appear to form distinct rodent-/shrew-borne clades within their respective genera, clustering separately from all currently classified viruses. In the case of the henipaviruses, a comparison of the different members of this clade revealed the presence of a secondary conserved open reading frame, encoding for a transmembrane protein, within the F gene, the biological relevance of which remains to be established. While the characteristics of the viruses described here shed further light on the complex evolutionary origin of paramyxoviruses, they also illustrate that the diversity of this group of viruses in terms of genome organization appears to be much larger than previously assumed.

Antimicrobial resistance analysis and whole-genome sequencing of Salmonella enterica serovar Indiana isolate from ducks

Salmonella is one of the most important genera of enteric pathogenic bacteria that threaten duck farms. The aim of this study was to increase the understanding of antimicrobial resistance mechanisms in Salmonella enterica serovar Indiana isolates of duck origin. Salmonella were isolated from duck cloacal swabs collected from duck farms located in Zhejiang and Henan provinces of China. All of the isolates were identified after a series of confirmatory tests including selective culture method, biochemical tests, serotyping and PCR targeting the invA gene. Isolates were then subjected to antimicrobial susceptibility testing by the standard Kirby-Bauer disk diffusion method. Subsequently, whole-genome sequencing analysis of a representative multidrug-resistant Salmonella Indiana isolate (designated SAP) was performed using a combination of Nanopore and Illumina sequencing platforms. A total of 18 Salmonella isolates were identified. The predominant serotype was Salmonella Indiana (14 of 18 isolates). All 14 Salmonella Indiana isolates were multiresistant to ten antimicrobials with multidrug resistance to ampicillin, cefoperazone, gentamicin, kanamycin, neomycin, tetracycline, norfloxacin, ciprofloxacin, colistin B and trimethoprim/sulfamethoxazole. The genome of Salmonella Indiana isolate SAP carried 65 antimicrobial resistance genes (ARGs) belonging to different families, including genes encoding antibiotic efflux pumps, rpsL, kdpDE, aac(6'), general bacterial porin with reduced permeability to β-lactams, AmpC-type β-lactamase gene, mutant lpx gene conferring resistance to colistin, sulfonamide-resistant dihydropteroate synthase folP and trimethoprim-resistant dihydrofolate reductase dfr. The Salmonella Indiana strain isolated in this study carried multiple ARGs and exhibited resistance to multiple antibiotics.

Chromosome-level genome assembly of the Chinese three-keeled pond turtle (Mauremys reevesii) provides insights into freshwater adaptation.

Mauremys reevesii is an endangered freshwater turtle that symbolizes longevity in Chinese culture. Despite its importance, genetic studies of this species remain limited, with no genomic sequence reported to date. Here, we report a high-quality, chromosome-level genomic sequence of M. reevesii obtained using a combination of Nanopore and Hi-C sequencing technologies. The 2.37Gb M. reevesii genome was assembled from a total of ~226.80Gb of Nanopore sequencing data. The M. reevesii genome contig N50 is 34.73Mb, the highest value in published turtle genomes. In total, 18,238genes were functionally annotated. The contigs were clustered and ordered onto 27pseudochromosomes covering ~96.55% of the genome assembled with Hi-C data. To explore genome evolution, synteny analysis was performed between M. reevesii (freshwater turtle) and Gopherus evgoodei (terrestrial turtle) genomes. In general, each chromosome of M. reevesii corresponded to one chromosome of Gopherus evgoodei, but some interchromosomal rearrangements occurred between the two species based on the assembled genomes. These interchromosomal rearrangements were further confirmed by mapping of the long-read nanopore data to the assembly. The reconstructed demographic history showed varied effective population size among freshwater, marine and terrestrial turtles. We also discovered expansion of genes related to the innate immune system in M. reevesii that may provide defence against freshwater pathogens. The high-quality genomic sequence provides a valuable genetic resource for further studies of genetics and genome evolution in turtles.

Genome Sequence Resource for Erysiphe necator NAFU1, a Grapevine Powdery Mildew Isolate Identified in Shaanxi Province of China.

Erysiphe necator is an economically important biotrophic fungal pathogen responsible for powdery mildew disease on grapevine. Currently, genome sequences are available for only a few E. necator isolates from the United States. Based on the combination of Nanopore and Illumina sequencing technologies, we present here the complete genome assembly for an isolate of E. necator, NAFU1, identified in China. We acquired a total of 15.93 Gb of raw reads. These reads were processed into a 61.12-Mb genome assembly containing 73 contigs with an N50 of 2.06 Mb and a maximum length of 6.05 Mb. Combining the results of three gene-prediction modules (i.e., an evidence-based gene modeler [EVidenceModeler], an ab initio gene modeler, and a homology-based gene modeler), we predicted 7,235 protein-coding genes in the assembled genome of E. necator NAFU1. This information will facilitate studies of genome evolution and pathogenicity mechanisms of E. necator and other powdery mildew species through comparative genome sequence analysis and other molecular genetic tools.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome Resources of Four Distinct Pathogenic Races Within Fusarium oxysporum f. sp. vasinfectum that Cause Vascular Wilt Disease of Cotton.

Whole genome sequence (WGS) based identifications are being increasingly used by regulatory and public health agencies to facilitate the detection, investigation, and control of pathogens and pests. Fusarium oxysporum f. sp. vasinfectum is a significant vascular wilt pathogen of cultivated cotton and consists of several pathogenic races that are not each other's closest phylogenetic relatives. We have developed WGS assemblies for isolates of F. oxysporum f. sp. vasinfectum race 1 (FOV1), race 4 (FOV4), race 5 (FOV5), and race 8 (FOV8) using a combination of Nanopore (MinION) and Illumina sequencing technology (Mi-Seq). This resulted in assembled contigs with more than 100× coverage for each of the F. oxysporum f. sp. vasinfectum races and estimated genome sizes of FOV1 52 Mb, FOV4 68 Mb, FOV5 68 Mb, and FOV8 55 Mb. The AUGUSTUS gene prediction program predicted 16,263 genes in FOV1, 20,259 genes in FOV4, 20,375 genes in FOV5 and 16,615 genes in FOV8. We were able to identify 525 genes unique to FOV1, 570 unique to FOV4, 1,242 unique to FOV5, and 383 unique to FOV8. We expect that these findings will help in comparative genomics and in the identification of unique genes as candidate targets for diagnostic marker and methods development to permit rapid differentiation of F. oxysporum f. sp. vasinfectum subgroups.

Sensing with Nanopores and Aptamers: A Way Forward.

In the 90s, the development of a novel single molecule technique based on nanopore sensing emerged. Preliminary improvements were based on the molecular or biological engineering of protein nanopores along with the use of nanotechnologies developed in the context of microelectronics. Since the last decade, the convergence between those two worlds has allowed for biomimetic approaches. In this respect, the combination of nanopores with aptamers, single-stranded oligonucleotides specifically selected towards molecular or cellular targets from an in vitro method, gained a lot of interest with potential applications for the single molecule detection and recognition in various domains like health, environment or security. The recent developments performed by combining nanopores and aptamers are highlighted in this review and some perspectives are drawn.

Complete Genome Sequence of a Virulent Leptospira interrogans Serovar Copenhageni Strain, Assembled with a Combination of Nanopore and Illumina Reads.

Here, we present the complete genome sequence of a highly virulent Leptospira interrogans serovar Copenhageni strain isolated from a dog with severe leptospirosis. In this work, a gapless genome draft was assembled with a combination of Nanopore and Illumina data of relatively low coverage.

Laser-induced fabrication of nanoporous monolayer WS2 membranes

Porous transition metal dichalcogenides (TMDs) are promising candidates for a variety of catalytic, purification, and energy storage applications. Despite recent advances, current fabrication techniques face issues concerning scalability and control over sample porosity. By utilizing water-assisted laser irradiation, we present here a new method for the fabrication of micron-scale, atomically-thin nanoporous tungsten disulfide (WS2) membranes. The electronic and physical structures of the porous membranes are characterized with photoluminescence (PL) spectroscopy and aberration-corrected scanning transmission electron microscopy (AC-STEM), respectively. With increasing laser irradiation dose, we observe a decay of PL signal, and a relative increase in the trion contribution compared to that of the neutral exciton, suggesting defect-related n-type doping and degradation of the membrane. AC-STEM images show the nucleation of tungsten oxide islands on the membrane, and the formation of triangular defect clusters containing a combination of nanopores and oxide-filled regions, providing insight at the atomic level into the photo-oxidation process in TMDs. A linear dependence of the nanoporous area percentage on the laser irradiation dose over the range of 102–105 W cm−2 is observed. The methods proposed here pave the way for the scalable production of nanoporous membranes through the laser-induced photo-oxidation of WS2 and other transition metal dichalcogenides.