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July 7, 2019

Complete genome sequence of Vibrio campbellii LMB 29 isolated from red drum with four native megaplasmids.

Vibrio spp. are the most common pathogens for animals reared in aquaculture. Vibrio campbellii, which is often involved in shrimp, fish and mollusks diseases, is widely distributed in the marine environment worldwide, but our knowledge about its pathogenesis and antimicrobial resistance is very limited. The existence of this knowledge gap is at least partially because that V. campbellii was originally classified as Vibrio harveyi, and the detailed information of its comparative genome analysis to other Vibrio spp. is currently lacking. In this study, the complete genome of a V. campbellii predominant strain, LMB29, was determined by MiSeq in conjunction with PacBio SMRT sequencing. This genome consists of two circular DNA chromosomes and four megaplasmids. Comparative genome analysis indicates that LMB29 shares a 96.66% similarity (average nucleotide identity) with the V. campbellii ATCC strain BAA-1116 based on a 75% AF (average fraction) calculations, and its functional profile is very similar to V. campbellii E1 and V. campbellii CAIM115. Both type III secretion system (T3SS) and type VI secretion system (T6SS), along with the tlh gene which encodes a thermolabile hemolysin, are present in LMB29 which may contribute to the bacterial pathogenesis. The virulence of this strain was experimental confirmed by performing a LDH assay on a fish cell infection model, and cell death was observed as early as within 3 h post infection. Thirty-seven antimicrobial resistance genes (>45% identity) were predicted in LMB29 which includes a novel rifampicin ADP ribosyltransferase, arr-9, in plasmid pLMB157. The gene arr-9 was predicted on a genomic island with horizontal transferable potentials which may facilitate the rifampicin resistance dissemination. Future researches are needed to explore the pathogenesis of V. campbellii LMB29, but the availability of this genome sequence will certainly aid as a basis for further analysis.

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July 7, 2019

Highly accurate fluorogenic DNA sequencing with information theory-based error correction.

Eliminating errors in next-generation DNA sequencing has proved challenging. Here we present error-correction code (ECC) sequencing, a method to greatly improve sequencing accuracy by combining fluorogenic sequencing-by-synthesis (SBS) with an information theory-based error-correction algorithm. ECC embeds redundancy in sequencing reads by creating three orthogonal degenerate sequences, generated by alternate dual-base reactions. This is similar to encoding and decoding strategies that have proved effective in detecting and correcting errors in information communication and storage. We show that, when combined with a fluorogenic SBS chemistry with raw accuracy of 98.1%, ECC sequencing provides single-end, error-free sequences up to 200 bp. ECC approaches should enable accurate identification of extremely rare genomic variations in various applications in biology and medicine.

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July 7, 2019

Global phylogenetic analysis of Escherichia coli and plasmids carrying the mcr-1 gene indicates bacterial diversity but plasmid restriction.

To understand the dynamics behind the worldwide spread of the mcr-1 gene, we determined the population structure of Escherichia coli and of mobile genetic elements (MGEs) carrying the mcr-1 gene. After a systematic review of the literature we included 65 E. coli whole genome sequences (WGS), adding 6 recently sequenced travel related isolates, and 312 MLST profiles. We included 219 MGEs described in 7 Enterobacteriaceae species isolated from human, animal and environmental samples. Despite a high overall diversity, 2 lineages were observed in the E. coli population that may function as reservoirs of the mcr-1 gene, the largest of which was linked to ST10, a sequence type known for its ubiquity in human faecal samples and in food samples. No genotypic clustering by geographical origin or isolation source was observed. Amongst a total of 13 plasmid incompatibility types, the IncI2, IncX4 and IncHI2 plasmids accounted for more than 90% of MGEs carrying the mcr-1 gene. We observed significant geographical clustering with regional spread of IncHI2 plasmids in Europe and IncI2 in Asia. These findings point towards promiscuous spread of the mcr-1 gene by efficient horizontal gene transfer dominated by a limited number of plasmid incompatibility types.

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July 7, 2019

N6-adenine DNA methylation is associated with the linker DNA of H2A.Z-containing well-positioned nucleosomes in Pol II-transcribed genes in Tetrahymena.

DNA N6-methyladenine (6mA) is newly rediscovered as a potential epigenetic mark across a more diverse range of eukaryotes than previously realized. As a unicellular model organism, Tetrahymena thermophila is among the first eukaryotes reported to contain 6mA modification. However, lack of comprehensive information about 6mA distribution hinders further investigations into its function and regulatory mechanism. In this study, we provide the first genome-wide, base pair-resolution map of 6mA in Tetrahymena by applying single-molecule real-time (SMRT) sequencing. We provide evidence that 6mA occurs mostly in the AT motif of the linker DNA regions. More strikingly, these linker DNA regions with 6mA are usually flanked by well-positioned nucleosomes and/or H2A.Z-containing nucleosomes. We also find that 6mA is exclusively associated with RNA polymerase II (Pol II)-transcribed genes, but is not an unambiguous mark for active transcription. These results support that 6mA is an integral part of the chromatin landscape shaped by adenosine triphosphate (ATP)-dependent chromatin remodeling and transcription.© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 7, 2019

DNA methylation profiling using long-read Single Molecule Real-Time bisulfite sequencing (SMRT-BS).

For the past two decades, bisulfite sequencing has been a widely used method for quantitative CpG methylation detection of genomic DNA. Coupled with PCR amplicon cloning, bisulfite Sanger sequencing allows for allele-specific CpG methylation assessment; however, its time-consuming protocol and inability to multiplex has recently been overcome by next-generation bisulfite sequencing techniques. Although high-throughput sequencing platforms have enabled greater accuracy in CpG methylation quantitation as a result of increased bisulfite sequencing depth, most common sequencing platforms generate reads that are similar in length to the typical bisulfite PCR size range (~300-500 bp). Using the Pacific Biosciences (PacBio) sequencing platform, we developed single molecule real-time bisulfite sequencing (SMRT-BS), which is an accurate targeted CpG methylation analysis method capable of a high degree of multiplexing and long read lengths. SMRT-BS is reproducible and was found to be concordant with other lower throughput quantitative CpG methylation methods. Moreover, the ability to sequence up to ~1.5-2.0 kb amplicons, when coupled with an optimized bisulfite-conversion protocol, allows for more thorough assessment of CpG islands and increases the capacity for studying the relationship between single nucleotide variants and allele-specific CpG methylation.

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July 7, 2019

pSY153-MDR, a p12969-DIM-related mega plasmid carrying blaIMP-45 and armA, from clinical Pseudomonas putida.

This work characterized mega plasmid pSY153-MDR, carrying blaIMP-45 and armA, from a multidrug-resistant (MDR) Pseudomonas putida isolate from the urine of a cerebral infarction patient in China. The backbone of pSY153-MDR was closely related to Pseudomonas plasmids p12969-DIM, pOZ176, pBM413, pTTS12, and pRBL16, and could not be assigned to any of the known incompatibility groups. The accessory modules of pSY153-MDR were composed of 10 individual insertion sequence elements and two different MDR regions, and differed dramatically from the above plasmids. Fifteen non-redundant resistance markers were identified to be involved in resistance to at least eight distinct classes of antibiotics. All of these resistance genes were associated with mobile elements, and were embedded within the two MDR regions. blaIMP-45 and armA coexisted in a Tn1403-Tn1548 region, which was generated from homologous recombination of Tn1403- and Tn1548-like transposons. The second copy of armA was a component of the ISCR28-armA-?ISCR28 structure, representing a novel armA vehicle. This vehicle was located within In48, which was related to In363 and In1058. Data presented here provide a deeper insight into the evolutionary history of SY153, especially in regard to how it became extensively drug-resistant.

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July 7, 2019

Comparative and population genomic landscape of Phellinus noxius: A hypervariable fungus causing root rot in trees.

The order Hymenochaetales of white rot fungi contain some of the most aggressive wood decayers causing tree deaths around the world. Despite their ecological importance and the impact of diseases they cause, little is known about the evolution and transmission patterns of these pathogens. Here, we sequenced and undertook comparative genomic analyses of Hymenochaetales genomes using brown root rot fungus Phellinus noxius, wood-decomposing fungus Phellinus lamaensis, laminated root rot fungus Phellinus sulphurascens and trunk pathogen Porodaedalea pini. Many gene families of lignin-degrading enzymes were identified from these fungi, reflecting their ability as white rot fungi. Comparing against distant fungi highlighted the expansion of 1,3-beta-glucan synthases in P. noxius, which may account for its fast-growing attribute. We identified 13 linkage groups conserved within Agaricomycetes, suggesting the evolution of stable karyotypes. We determined that P. noxius has a bipolar heterothallic mating system, with unusual highly expanded ~60 kb A locus as a result of accumulating gene transposition. We investigated the population genomics of 60 P. noxius isolates across multiple islands of the Asia Pacific region. Whole-genome sequencing showed this multinucleate species contains abundant poly-allelic single nucleotide polymorphisms with atypical allele frequencies. Different patterns of intra-isolate polymorphism reflect mono-/heterokaryotic states which are both prevalent in nature. We have shown two genetically separated lineages with one spanning across many islands despite the geographical barriers. Both populations possess extraordinary genetic diversity and show contrasting evolutionary scenarios. These results provide a framework to further investigate the genetic basis underlying the fitness and virulence of white rot fungi.© 2017 John Wiley & Sons Ltd.

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July 7, 2019

Filling reference gaps via assembling DNA barcodes using high-throughput sequencing-moving toward barcoding the world.

Over the past decade, biodiversity researchers have dedicated tremendous efforts to constructing DNA reference barcodes for rapid species registration and identification. Although analytical cost for standard DNA barcoding has been significantly reduced since early 2000, further dramatic reduction in barcoding costs is unlikely because Sanger sequencing is approaching its limits in throughput and chemistry cost. Constraints in barcoding cost not only led to unbalanced barcoding efforts around the globe, but also prevented high-throughput sequencing (HTS)-based taxonomic identification from applying binomial species names, which provide crucial linkages to biological knowledge. We developed an Illumina-based pipeline, HIFI-Barcode, to produce full-length Cytochrome c oxidase subunit I (COI) barcodes from pooled polymerase chain reaction amplicons generated by individual specimens. The new pipeline generated accurate barcode sequences that were comparable to Sanger standards, even for different haplotypes of the same species that were only a few nucleotides different from each other. Additionally, the new pipeline was much more sensitive in recovering amplicons at low quantity. The HIFI-Barcode pipeline successfully recovered barcodes from more than 78% of the polymerase chain reactions that didn’t show clear bands on the electrophoresis gel. Moreover, sequencing results based on the single molecular sequencing platform Pacbio confirmed the accuracy of the HIFI-Barcode results. Altogether, the new pipeline can provide an improved solution to produce full-length reference barcodes at about one-tenth of the current cost, enabling construction of comprehensive barcode libraries for local fauna, leading to a feasible direction for DNA barcoding global biomes.© The Authors 2017. Published by Oxford University Press.

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July 7, 2019

Comparative analysis of the radish genome with Brassica genomes

Raphanus sativus L. includes an annual root vegetable crop, radish, and diverse wild species. R. sativus has a long history of domestication, but its phylogenetic position in the tribe Brassiceae is controversial. A comprehensive analysis of the R. sativus genome will provide fundamental information about the structure of its genome, evolutionary features of polyploidy, and significant insight for phylogenetic delimitation of this species. Diverse genomic resources, including a high-density genetic map, clone libraries, cytogenetic data, and transcriptome data, have been developed to sequence the genome. Recently, the R. sativus cv. ‘WK10039’ (2n = 18, 510.8 Mb) genome was sequenced and assembled into nine chromosome pseudomolecules spanning >98% of the gene space. Comparative mapping of the tPCK-like ancestral genome based on conserved ortholog set markers and proteome comparison revealed that the R. sativus genome has intermediate characteristics between the Brassica A/C and B genomes with triplicated segments, suggesting an internal origin from the genus Brassica. The evolutionary characteristics shared between R. sativus and diploid Brassica species provide genomic evidence for species delimitation of R. sativus and reconstruction of the mesohexaploid ancestral genome.

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July 7, 2019

Genomics of parallel adaptation at two timescales in Drosophila.

Two interesting unanswered questions are the extent to which both the broad patterns and genetic details of adaptive divergence are repeatable across species, and the timescales over which parallel adaptation may be observed. Drosophila melanogaster is a key model system for population and evolutionary genomics. Findings from genetics and genomics suggest that recent adaptation to latitudinal environmental variation (on the timescale of hundreds or thousands of years) associated with Out-of-Africa colonization plays an important role in maintaining biological variation in the species. Additionally, studies of interspecific differences between D. melanogaster and its sister species D. simulans have revealed that a substantial proportion of proteins and amino acid residues exhibit adaptive divergence on a roughly few million years long timescale. Here we use population genomic approaches to attack the problem of parallelism between D. melanogaster and a highly diverged conger, D. hydei, on two timescales. D. hydei, a member of the repleta group of Drosophila, is similar to D. melanogaster, in that it too appears to be a recently cosmopolitan species and recent colonizer of high latitude environments. We observed parallelism both for genes exhibiting latitudinal allele frequency differentiation within species and for genes exhibiting recurrent adaptive protein divergence between species. Greater parallelism was observed for long-term adaptive protein evolution and this parallelism includes not only the specific genes/proteins that exhibit adaptive evolution, but extends even to the magnitudes of the selective effects on interspecific protein differences. Thus, despite the roughly 50 million years of time separating D. melanogaster and D. hydei, and despite their considerably divergent biology, they exhibit substantial parallelism, suggesting the existence of a fundamental predictability of adaptive evolution in the genus.

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July 7, 2019

Genetic maps and whole genome sequences of radish

Radish, Raphanus sativus L., is a member of Brassicaceae, to which Arabidopsis thaliana, a model plant in plant biology, belongs, as do other Brassica species including important crops. However, genetic and genomic studies of radish have been behind those of Arabidopsis and Brassica. In this decade, much effort has been made to develop genetic resources for radish, e.g., DNA markers, genetic maps, and whole genome sequences. Studies using the obtained information have revealed the genome structure of radish in terms of ancestral karyotype and have also prompted the identification of genes for agronomically important traits in radish through a map-based cloning strategy and quantitative trait locus analysis. In this chapter, we review the evolving development of radish genetic map in the past 15 years and the current status of genome sequencing of radish. We also introduce the latest strategy for the construction of a high-density genetic map using next-generation sequencing technology and propose a prospective direction of genetics and genomics research in radish which would be helpful for researchers and breeders in their efforts to promote radish breeding programs efficiently.

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July 7, 2019

Complete genome sequence analysis of Enterobacter sp. SA187, a plant multi-stress tolerance promoting endophytic bacterium

Enterobacter sp. SA187 is an endophytic bacterium that has been isolated from root nodules of the indigenous desert plant Indigofera argentea. SA187 could survive in the rhizosphere as well as in association with different plant species, and was able to provide abiotic stress tolerance to Arabidopsis thaliana. The genome sequence of SA187 was obtained by using Pacific BioScience (PacBio) single-molecule sequencing technology, with average coverage of 275X. The genome of SA187 consists of one single 4,429,597 bp chromosome, with an average 56% GC content and 4,347 predicted protein coding DNA sequences (CDS), 153 ncRNA, 7 rRNA, and 84 tRNA. Functional analysis of the SA187 genome revealed a large number of genes involved in uptake and exchange of nutrients, chemotaxis, mobilization and plant colonization. A high number of genes were also found to be involved in survival, defense against oxidative stress and production of antimicrobial compounds and toxins. Moreover, different metabolic pathways were identified that potentially contribute to plant growth promotion. The information encoded in the genome of SA187 reveals the characteristics of a dualistic lifestyle of a bacterium that can adapt to different environments and promote the growth of plants. This information provides a better understanding of the mechanisms involved in plant-microbe interaction and could be further exploited to develop SA187 as a biological agent to improve agricultural practices in marginal and arid lands.

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July 7, 2019

Remarkable diversity of Escherichia coli carrying mcr-1 from hospital sewage with the identification of two new mcr-1 variants.

The plasmid-borne colistin-resistant gene mcr-1 has rapidly become a worldwide public health concern. This study aims to determine the host bacterial strains, plasmids, and genetic contexts of mcr-1 in hospital sewage. A 1-ml hospital sewage sample was cultured. Colistin-resistant bacterial colonies were selected on agar plates and were subjected to whole genome sequencing and subsequent analysis. The transfer of mcr-1 between bacterial strains was tested using conjugation. New variants of mcr-1 were cloned to test the impact of variations on the function of mcr-1. Plasmids carrying mcr-1 were retrieved from GenBank for comparison based on concatenated backbone genes. In the sewage sample, we observed that mcr-1 was located in various genetic contexts on the chromosome, or plasmids of four different replicon types (IncHI2, IncI2, IncP, and IncX4), in Klebsiella pneumoniae, Kluyvera spp. and seven Escherichia coli strains of six different sequence types (ST10, ST34, ST48, ST1196, ST7086, and ST7087). We also identified two new variants of mcr-1, mcr-1.4 and mcr-1.7, both of which encode an amino acid variation from mcr-1. mcr-1-carrying IncX4 plasmids, which have a global distribution across the Enterobacteriaceae, are the result of global dissemination of a single common plasmid, while IncI2 mcr-1 plasmids appear to acquire mcr-1 in multiple events. In conclusion, the unprecedented remarkable diversity of species, strains, plasmids, and genetic contexts carrying mcr-1 present in a single sewage sample from a single healthcare site highlights the continued evolution and dynamic transmission of mcr-1 in healthcare-associated environments.

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July 7, 2019

Molecular approaches for high throughput detection and quantification of genetically modified crops: A review.

As long as the genetically modified crops are gaining attention globally, their proper approval and commercialization need accurate and reliable diagnostic methods for the transgenic content. These diagnostic techniques are mainly divided into two major groups, i.e., identification of transgenic (1) DNA and (2) proteins from GMOs and their products. Conventional methods such as PCR (polymerase chain reaction) and enzyme-linked immunosorbent assay (ELISA) were routinely employed for DNA and protein based quantification respectively. Although, these Techniques (PCR and ELISA) are considered as significantly convenient and productive, but there is need for more advance technologies that allow for high throughput detection and the quantification of GM event as the production of more complex GMO is increasing day by day. Therefore, recent approaches like microarray, capillary gel electrophoresis, digital PCR and next generation sequencing are more promising due to their accuracy and precise detection of transgenic contents. The present article is a brief comparative study of all such detection techniques on the basis of their advent, feasibility, accuracy, and cost effectiveness. However, these emerging technologies have a lot to do with detection of a specific event, contamination of different events and determination of fusion as well as stacked gene protein are the critical issues to be addressed in future.

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