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Asset Tag: Third generation sequencing

April 21, 2020

Single-molecule sequencing detection of N6-methyladenine in microbial reference materials.

The DNA base modification N6-methyladenine (m6A) is involved in many pathways related to the survival of bacteria and their interactions with hosts. Nanopore sequencing offers a new, portable method to detect base modifications. Here, we show that a neural network can improve m6A detection at trained sequence contexts compared to previously published methods using deviations between measured and expected current values as each adenine travels through a pore. The model, implemented as the mCaller software package, can be extended to detect known or confirm suspected methyltransferase target motifs based on predictions of methylation at untrained contexts. We use PacBio, Oxford Nanopore, methylated DNA immunoprecipitation sequencing (MeDIP-seq), and whole-genome bisulfite sequencing data to generate and orthogonally validate methylomes for eight microbial reference species. These well-characterized microbial references can serve as controls in the development and evaluation of future methods for the identification of base modifications from single-molecule sequencing data.

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April 21, 2020

A Phage-Like Plasmid Carrying blaKPC-2 Gene in Carbapenem-Resistant Pseudomonas aeruginosa.

Background: Lateral gene transfer plays a central role in the dissemination of carbapenem resistance in bacterial pathogens associated with nosocomial infections, mainly Enterobacteriaceae and Pseudomonas aeruginosa. Despite their clinical significance, there is little information regarding the mobile genetic elements and mechanism of acquisition and propagation of lateral genes in P. aeruginosa, and they remain largely unknown. Objectives: The present study characterized the genetic context of blaKPC-2 in carbapenem-resistant P. aeruginosa strain BH9. Methods:Pseudomonas aeruginosa BH9 sequencing was performed using the long-read PacBio SMRT platform and the Ion Proton System. De novo assembly was carried out using the SMRT pipeline and Canu, and gene prediction and annotation were performed using Prokka and RAST. Results:Pseudomonas aeruginosa BH9 exhibited a 7.1 Mb circular chromosome. However, the blaKPC-2 gene is located in an additional contig composed by a small plasmid pBH6 from P. aeruginosa strain BH6 and several phage-related genes. Further analysis revealed that the beginning and end of the contig contain identical sequences, supporting a circular plasmid structure. This structure spans 41,087 bp, exhibiting all the Mu-like phage landmarks. In addition, 5-bp direct repeats (GGATG) flanking the pBH6 ends were found, strongly indicating integration of the Mu-like phage into the pBH6 plasmid. Mu phages are commonly found in P. aeruginosa. However, for the first time showing a potential impact in shaping the vehicles of the dissemination of antimicrobial (e.g., plasmid pBH6) resistance genes in the Pseudomonas genus. Conclusion: pBH6 captured the Mu-like Phage BH9, creating a co-integrate pBH6::Phage BH9, and this phage-plasmid complex may represent novel case of a phage-like plasmid.

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April 21, 2020

Reference genome and comparative genome analysis for the WHO reference strain for Mycobacterium bovis BCG Danish, the present tuberculosis vaccine.

Mycobacterium bovis bacillus Calmette-Guérin (M. bovis BCG) is the only vaccine available against tuberculosis (TB). In an effort to standardize the vaccine production, three substrains, i.e. BCG Danish 1331, Tokyo 172-1 and Russia BCG-1 were established as the WHO reference strains. Both for BCG Tokyo 172-1 as Russia BCG-1, reference genomes exist, not for BCG Danish. In this study, we set out to determine the completely assembled genome sequence for BCG Danish and to establish a workflow for genome characterization of engineering-derived vaccine candidate strains.By combining second (Illumina) and third (PacBio) generation sequencing in an integrated genome analysis workflow for BCG, we could construct the completely assembled genome sequence of BCG Danish 1331 (07/270) (and an engineered derivative that is studied as an improved vaccine candidate, a SapM KO), including the resolution of the analytically challenging long duplication regions. We report the presence of a DU1-like duplication in BCG Danish 1331, while this tandem duplication was previously thought to be exclusively restricted to BCG Pasteur. Furthermore, comparative genome analyses of publicly available data for BCG substrains showed the absence of a DU1 in certain BCG Pasteur substrains and the presence of a DU1-like duplication in some BCG China substrains. By integrating publicly available data, we provide an update to the genome features of the commonly used BCG strains.We demonstrate how this analysis workflow enables the resolution of genome duplications and of the genome of engineered derivatives of the BCG Danish vaccine strain. The BCG Danish WHO reference genome will serve as a reference for future engineered strains and the established workflow can be used to enhance BCG vaccine standardization.

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April 21, 2020

Systematic identification of intergenic long-noncoding RNAs in mouse retinas using full-length isoform sequencing.

A great mass of long noncoding RNAs (lncRNAs) have been identified in mouse genome and increasing evidences in the last decades have revealed their crucial roles in diverse biological processes. Nevertheless, the biological roles of lncRNAs in the mouse retina remains largely unknown due to the lack of a comprehensive annotation of lncRNAs expressed in the retina.In this study, we applied the long-reads sequencing strategy to unravel the transcriptomes of developing mouse retinas and identified a total of 940 intergenic lncRNAs (lincRNAs) in embryonic and neonatal retinas, including about 13% of them were transcribed from unannotated gene loci. Subsequent analysis revealed that function of lincRNAs expressed in mouse retinas were closely related to the physiological roles of this tissue, including 90 lincRNAs that were differentially expressed after the functional loss of key regulators of retinal ganglion cell (RGC) differentiation. In situ hybridization results demonstrated the enrichment of three class IV POU-homeobox genes adjacent lincRNAs (linc-3a, linc-3b and linc-3c) in ganglion cell layer and indicated they were potentially RGC-specific.In summary, this study systematically annotated the lincRNAs expressed in embryonic and neonatal mouse retinas and implied their crucial regulatory roles in retinal development such as RGC differentiation.

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April 21, 2020

Methicillin-Resistant Staphylococcus aureus Blood Isolates Harboring a Novel Pseudo-staphylococcal Cassette Chromosome mec Element.

The aim of this work was to assess a novel pseudo-staphylococcal cassette chromosome mec (?SCCmec) element in methicillin-resistant Staphylococcus aureus (MRSA) blood isolates. Community-associated MRSA E16SA093 and healthcare-associated MRSA F17SA003 isolates were recovered from the blood specimens of patients with S. aureus bacteremia in 2016 and in 2017, respectively. Antimicrobial susceptibility was determined via the disk diffusion method, and SCCmec typing was conducted by multiplex polymerase chain reaction. Whole genome sequencing was carried out by single molecule real-time long-read sequencing. Both isolates belonged to sequence type 72 and agr-type I, and they were negative for Panton-Valentine leukocidin and toxic shock syndrome toxin. The spa-types of E16SA093 and F17SA003 were t324 and t2460, respectively. They had a SCCmec IV-like element devoid of the cassette chromosome recombinase (ccr) gene complex, designated as ?SCCmecE16SA093. The element was manufactured from SCCmec type IV and the deletion of the ccr gene complex and a 7.0- and 31.9-kb portion of each chromosome. The deficiency of the ccr gene complex in the SCCmec unit is likely resulting in mobility loss, which would be an adaptive evolutionary mechanism. The dissemination of this clone should be monitored closely.

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April 21, 2020

A Pathovar of Xanthomonas oryzae Infecting Wild Grasses Provides Insight Into the Evolution of Pathogenicity in Rice Agroecosystems

Xanthomonas oryzae (Xo) are critical rice pathogens. Virulent lineages from Africa and Asia and less virulent strains from the US have been well characterized. X. campestris pv. leersiae (Xcl), first described in 1957, causes bacterial streak on the perennial grass, Leersia hexandra, and is a close relative of Xo. L. hexandra, a member of the Poaceae, is highly similar to rice phylogenetically, is globally ubiquitous around rice paddies, and is a reservoir of pathogenic Xo. We used long read, single molecule, real time (SMRT) genome sequences of five strains of Xcl from Burkina Faso, China, Mali and Uganda to determine the genetic relatedness of this organism with Xo. Novel Transcription Activator-Like Effectors (TALEs) were discovered in all five strains of Xcl. Predicted TALE target sequences were identified in the L. perrieri genome and compared to rice susceptibility gene homologs. Pathogenicity screening on L. hexandra and diverse rice cultivars confirmed that Xcl are able to colonize rice and produce weak but not progressive symptoms. Overall, based on average nucleotide identity, type III effector repertoires and disease phenotype, we propose to rename Xcl to X. oryzae pv. leersiae (Xol) and use this parallel system to improve understanding of the evolution of bacterial pathogenicity in rice agroecosystems.

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April 21, 2020

Prediction of Host-Specific Genes by Pan-Genome Analyses of the Korean Ralstonia solanacearum Species Complex.

The soil-borne pathogenic Ralstonia solanacearum species complex (RSSC) is a group of plant pathogens that is economically destructive worldwide and has a broad host range, including various solanaceae plants, banana, ginger, sesame, and clove. Previously, Korean RSSC strains isolated from samples of potato bacterial wilt were grouped into four pathotypes based on virulence tests against potato, tomato, eggplant, and pepper. In this study, we sequenced the genomes of 25 Korean RSSC strains selected based on these pathotypes. The newly sequenced genomes were analyzed to determine the phylogenetic relationships between the strains with average nucleotide identity values, and structurally compared via multiple genome alignment using Mauve software. To identify candidate genes responsible for the host specificity of the pathotypes, functional genome comparisons were conducted by analyzing pan-genome orthologous group (POG) and type III secretion system effectors (T3es). POG analyses revealed that a total of 128 genes were shared only in tomato-non-pathogenic strains, 8 genes in tomato-pathogenic strains, 5 genes in eggplant-non-pathogenic strains, 7 genes in eggplant-pathogenic strains, 1 gene in pepper-non-pathogenic strains, and 34 genes in pepper-pathogenic strains. When we analyzed T3es, three host-specific effectors were predicted: RipS3 (SKWP3) and RipH3 (HLK3) were found only in tomato-pathogenic strains, and RipAC (PopC) were found only in eggplant-pathogenic strains. Overall, we identified host-specific genes and effectors that may be responsible for virulence functions in RSSC in silico. The expected characters of those genes suggest that the host range of RSSC is determined by the comprehensive actions of various virulence factors, including effectors, secretion systems, and metabolic enzymes.

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April 21, 2020

Adaptations of Alteromonas sp. 76-1 to Polysaccharide Degradation: A CAZyme Plasmid for Ulvan Degradation and Two Alginolytic Systems.

Studying the physiology and genomics of cultured hydrolytic bacteria is a valuable approach to decipher the biogeochemical cycling of marine polysaccharides, major nutrients derived from phytoplankton and macroalgae. We herein describe the profound potential of Alteromonas sp. 76-1, isolated from alginate-enriched seawater at the Patagonian continental shelf, to degrade the algal polysaccharides alginate and ulvan. Phylogenetic analyses indicated that strain 76-1 might represent a novel species, distinguished from its closest relative (Alteromonas naphthalenivorans) by adaptations to their contrasting habitats (productive open ocean vs. coastal sediments). Ecological distinction of 76-1 was particularly manifested in the abundance of carbohydrate-active enzymes (CAZymes), consistent with its isolation from alginate-enriched seawater and elevated abundance of a related OTU in the original microcosm. Strain 76-1 encodes multiple alginate lyases from families PL6, PL7, PL17, and PL18 largely contained in two polysaccharide utilization loci (PUL), which may facilitate the utilization of different alginate structures in nature. Notably, ulvan degradation relates to a 126 Kb plasmid dedicated to polysaccharide utilization, encoding several PL24 and PL25 ulvan lyases and monomer-processing genes. This extensive and versatile CAZyme repertoire allowed substantial growth on polysaccharides, showing comparable doubling times with alginate (2 h) and ulvan (3 h) in relation to glucose (3 h). The finding of homologous ulvanolytic systems in distantly related Alteromonas spp. suggests CAZyme plasmids as effective vehicles for PUL transfer that mediate niche gain. Overall, the demonstrated CAZyme repertoire substantiates the role of Alteromonas in marine polysaccharide degradation and how PUL exchange influences the ecophysiology of this ubiquitous marine taxon.

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April 21, 2020

Closing the Yield Gap for Cannabis: A Meta-Analysis of Factors Determining Cannabis Yield.

Until recently, the commercial production of Cannabis sativa was restricted to varieties that yielded high-quality fiber while producing low levels of the psychoactive cannabinoid tetrahydrocannabinol (THC). In the last few years, a number of jurisdictions have legalized the production of medical and/or recreational cannabis with higher levels of THC, and other jurisdictions seem poised to follow suit. Consequently, demand for industrial-scale production of high yield cannabis with consistent cannabinoid profiles is expected to increase. In this paper we highlight that currently, projected annual production of cannabis is based largely on facility size, not yield per square meter. This meta-analysis of cannabis yields reported in scientific literature aimed to identify the main factors contributing to cannabis yield per plant, per square meter, and per W of lighting electricity. In line with previous research we found that variety, plant density, light intensity and fertilization influence cannabis yield and cannabinoid content; we also identified pot size, light type and duration of the flowering period as predictors of yield and THC accumulation. We provide insight into the critical role of light intensity, quality, and photoperiod in determining cannabis yields, with particular focus on the potential for light-emitting diodes (LEDs) to improve growth and reduce energy requirements. We propose that the vast amount of genomics data currently available for cannabis can be used to better understand the effect of genotype on yield. Finally, we describe diversification that is likely to emerge in cannabis growing systems and examine the potential role of plant-growth promoting rhizobacteria (PGPR) for growth promotion, regulation of cannabinoid biosynthesis, and biocontrol.

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April 21, 2020

Virulence characteristics and an action mode of antibiotic resistance in multidrug-resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa displays intrinsic resistance to many antibiotics and known to acquire actively genetic mutations for further resistance. In this study, we attempted to understand genomic and transcriptomic landscapes of P. aeruginosa clinical isolates that are highly resistant to multiple antibiotics. We also aimed to reveal a mode of antibiotic resistance by elucidating transcriptional response of genes conferring antibiotic resistance. To this end, we sequenced the whole genomes and profiled genome-wide RNA transcripts of three different multi-drug resistant (MDR) clinical isolates that are phylogenetically distant from one another. Multi-layered genome comparisons with genomes of antibiotic-susceptible P. aeruginosa strains and 70 other antibiotic-resistance strains revealed both well-characterized conserved gene mutations and distinct distribution of antibiotic-resistant genes (ARGs) among strains. Transcriptions of genes involved in quorum sensing and type VI secretion systems were invariably downregulated in the MDR strains. Virulence-associated phenotypes were further examined and results indicate that our MDR strains are clearly avirulent. Transcriptions of 64 genes, logically selected to be related with antibiotic resistance in MDR strains, were active under normal growth conditions and remained unchanged during antibiotic treatment. These results propose that antibiotic resistance is achieved by a “constitutive” response scheme, where ARGs are actively expressed even in the absence of antibiotic stress, rather than a “reactive” response. Bacterial responses explored at the transcriptomic level in conjunction with their genome repertoires provided novel insights into (i) the virulence-associated phenotypes and (ii) a mode of antibiotic resistance in MDR P. aeruginosa strains.

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April 21, 2020

Directed Repeats Co-occur with Few Short-Dispersed Repeats in Plastid Genome of a Spikemoss, Selaginella vardei (Selaginellaceae, Lycopodiopsida).

It is hypothesized that the highly conserved inverted repeats (IR) structure of land plant plastid genomes (plastomes) is beneficial for stabilizing plastome organization, whereas the mechanism of the occurrence and stability maintenance of the recently reported direct repeats (DR) structure is yet awaiting further exploration. Here we describe the DR structure of the Selaginella vardei (Selaginellaceae) plastome, to elucidate the mechanism of DR occurrence and stability maintenance.The plastome of S. vardei is 121,254 bp in length and encodes 76 genes, of which 62 encode proteins, 10 encode tRNAs, and four encode rRNAs. Unexpectedly, the two identical rRNA gene regions (13,893 bp) are arranged in a direct orientation (DR), rather than inverted. Comparing to the IR organization in Isoetes flaccida (Isoetaceae, Lycopodiopsida) plastome, a ca. 50-kb trnN-trnF inversion that spans one DR copy was found in the plastome of S. vardei, which might cause the orientation change. In addition, we find extremely rare short dispersed repeats (SDRs) in the plastomes of S. vardei and its closely related species S. indica.We suggest that the ca. 50-kb inversion resulted in the DR structure, and the reduction in SDRs plays a key role in maintaining the stability of plastomes with DR structure by avoiding potential secondary recombination. We further confirmed the presence of homologous recombination between DR regions, which are able to generate subgenomes and form diverse multimers. Our study deepens the understanding of Selaginella plastomes and provides new insights into the diverse plastome structures in land plants.

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April 21, 2020

A genomic resource derived from the integration of genome sequences, expressed transcripts and genetic markers in ramie.

The redundancy of genomic resources, including transcript and molecular markers, and their uncertain position in the genome have dramatically hindered the study of traits in ramie, an important natural fiber crop.We obtained a high-quality transcriptome consisting of 30,591 non-redundant transcripts using single-molecule long-read sequencing and proposed it as a universal ramie transcriptome. Additionally, 55,882 single nucleotide polymorphisms (SNPs) were identified and a high-density genetic map was developed. Based on this genetic map, 181.7?Mb ramie genome sequences were assembled into 14 chromosomes. For the convenient use of these resources, 29,286 (~?95.7%) of the transcripts and all 55,882 SNPs, along with 1827 previously reported sequence repeat markers (SSRs), were mapped into the ramie genome, and 22,343 (~?73.0%) transcripts, 50,154 (~?89.7%) SNPs, and 1466 (~?80.3%) SSRs were assigned to a specific location in the corresponding chromosome.This is the first study to characterize the ramie transcriptome by long-read sequencing, and the substantial number of transcripts of significant length obtained will accelerate our understanding of ramie growth and development. This integration of genome sequences, expressed transcripts, and genetic markers will provide an extremely useful resource for genetic, molecular, and breeding studies of ramie.

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April 21, 2020

Genomic erosion and extensive horizontal gene transfer in gut-associated Acetobacteraceae.

Symbiotic relationships between animals and bacteria have profound impacts on the evolutionary trajectories of each partner. Animals and gut bacteria engage in a variety of relationships, occasionally persisting over evolutionary timescales. Ants are a diverse group of animals that engage in many types of associations with taxonomically distinct groups of bacterial associates. Here, we bring into culture and characterize two closely-related strains of gut associated Acetobacteraceae (AAB) of the red carpenter ant, Camponotus chromaiodes.Genome sequencing, assembly, and annotation of both strains delineate stark patterns of genomic erosion and sequence divergence in gut associated AAB. We found widespread horizontal gene transfer (HGT) in these bacterial associates and report elevated gene acquisition associated with energy production and conversion, amino acid and coenzyme transport and metabolism, defense mechanisms, and lysine export. Both strains have acquired the complete NADH-quinone oxidoreductase complex, plausibly from an Enterobacteriaceae origin, likely facilitating energy production under diverse conditions. Conservation of several lysine biosynthetic and salvage pathways and accumulation of lysine export genes via HGT implicate L-lysine supplementation by both strains as a potential functional benefit for the host. These trends are contrasted by genome-wide erosion of several amino acid biosynthetic pathways and pathways in central metabolism. We perform phylogenomic analyses on both strains as well as several free living and host associated AAB. Based on their monophyly and deep divergence from other AAB, these C. chromaiodes gut associates may represent a novel genus. Together, our results demonstrate how extensive horizontal transfer between gut associates along with genome-wide deletions leads to mosaic metabolic pathways. More broadly, these patterns demonstrate that HGT and genomic erosion shape metabolic capabilities of persistent gut associates and influence their genomic evolution.Using comparative genomics, our study reveals substantial changes in genomic content in persistent associates of the insect gastrointestinal tract and provides evidence for the evolutionary pressures inherent to this environment. We describe patterns of genomic erosion and horizontal acquisition that result in mosaic metabolic pathways. Accordingly, the phylogenetic position of both strains of these associates form a divergent, monophyletic clade sister to gut associates of honey bees and more distantly to Gluconobacter.

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April 21, 2020

Chromosome conformation capture resolved near complete genome assembly of broomcorn millet.

Broomcorn millet (Panicum miliaceum L.) has strong tolerance to abiotic stresses, and is probably one of the oldest crops, with its earliest cultivation that dated back to ca. ~10,000 years. We report here its genome assembly through a combination of PacBio sequencing, BioNano, and Hi-C (in vivo) mapping. The 18 super scaffolds cover ~95.6% of the estimated genome (~887.8?Mb). There are 63,671 protein-coding genes annotated in this tetraploid genome. About ~86.2% of the syntenic genes in foxtail millet have two homologous copies in broomcorn millet, indicating rare gene loss after tetraploidization in broomcorn millet. Phylogenetic analysis reveals that broomcorn millet and foxtail millet diverged around ~13.1 Million years ago (Mya), while the lineage specific tetraploidization of broomcorn millet may be happened within ~5.91 million years. The genome is not only beneficial for the genome assisted breeding of broomcorn millet, but also an important resource for other Panicum species.

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April 21, 2020

Genome-wide profiling of the alternative splicing provides insights into development in Plutella xylostella.

The diamondback moth (DBM), Plutella xylostella (L.), is a major pest of cruciferous crops worldwide. While the species has become a model for genomics, post-transcriptional mechanisms associated with development and sex determination have not been comprehensively studied and the lack of complete structure of mRNA transcripts limits further research.Here, we combined the methods of single-molecule long-read sequencing technology (IsoSeq) and RNA-seq to re-annotate the published DBM genome and present the genome-wide identification of alternative splicing (AS) associated with development and sex determination of DBM. In total, we identified ~?13,900 genes (~?77%) annotated in the DBM genome (version-2), resulting in the correction of 1586 wrongly annotated genes and identification of 78,000 previously unannotated transcripts. We also identified 1804 genes showing alternative splicing (AS) in each of the developmental stages and sexes, suggesting that AS events are ubiquitous in DBM. Comparative analyses showed that these AS events were rarely shared among developmental stages, indicating that they may play key specific roles in regulation of insect development. Further, we found 156 genes showing different AS events and expression patterns between males and females, linking them to potential functions in sex determination.Overall, the P. xylostella transcriptome provides the significant information about regulatory alternative splicing events, which are shown to be involved in development and sex determination. Our work presents a solid foundation to better understand the mechanism of post-transcriptional regulation, and offers wider insights into insect development and sex determination.

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