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April 21, 2020

Genome sequence resource for Ilyonectria mors-panacis, causing rusty root rot of Panax notoginseng.

Ilyonectria mors-panacis is a serious disease hampering the production of Panax notoginseng, an important Chinese medicinal herb, widely used for its anti-inflammatory, anti-fatigue, hepato-protective, and coronary heart disease prevention effects. Here, we report the first Illumina-Pacbio hybrid sequenced draft genome assembly of I. mors-panacis strain G3B and its annotation. The availability of this genome sequence not only represents an important tool toward understanding the genetics behind the infection mechanism of I. mors-panacis strain G3B but also will help illuminate the complexities of the taxonomy of this species.

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April 21, 2020

Genomics-informed molecular detection of Xanthomonas vasicola pv. vasculorum strains causing severe bacterial leaf streak of corn.

Xanthomonas vasicola pv. vasculorum (syn. X. campestris pv. vasculorum) was initially identified as the causal agent of bacterial leaf streak of corn in South Africa. The pathovar vasculorum causes disease on sugarcane and corn, but a subset of these strains was noted for its increased disease severity in corn. This subset was re-classified as Xanthomonas campestris pv. zeae in the early 1990s and was found to have slightly different biochemical and genetic properties than isolates from sugarcane. There has been an emergence of X. campestris pv. zeae-like strains of X. vasicola pv. vasculorum in both the United States and Argentina since 2010. We performed whole genome sequencing on U.S. isolates to confirm their identity. Informed by comparative genomics, we then developed specific TaqMan qPCR and loop-mediated isothermal amplification (LAMP) assays for the detection of this specific subset of X. vasicola pv. vasculorum strains. The qPCR 4909 assay was tested against 27 xanthomonads (diverse representation), 32 DNA extractions from corn leaves confirmed as positive or negative for the bacterium, 41 X. vasicola pv. vasculorum isolates from corn in the United States and Argentina, and 31 additional bacteria associated with corn, sugarcane, or sorghum. In all cases the assay was shown to be specific for the X. vasicola pv. vasculorum isolates that cause more severe disease on corn. We then tested the LAMP 166 assay against the 27 xanthomonads and 32 corn leaf DNA samples, and we found this assay was also specific for this subset of X. vasicola pv. vasculorum isolates. We also developed a live/dead cells distinction protocol using propidium monoazide prior to DNA extraction for analyzing seed washes using these assays. These two detection assays can be useful for both diagnosticians and researchers to specifically identify the X. vasicola pv. vasculorum isolates that cause more severe symptoms on corn.

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April 21, 2020

Next-Generation Sequencing and Emerging Technologies.

Genetic sequencing technologies are evolving at a rapid pace with major implications for research and clinical practice. In this review, the authors provide an updated overview of next-generation sequencing (NGS) and emerging methodologies. NGS has tremendously improved sequencing output while being more time and cost-efficient in comparison to Sanger sequencing. The authors describe short-read sequencing approaches, such as sequencing by synthesis, ion semiconductor sequencing, and nanoball sequencing. Third-generation long-read sequencing now promises to overcome many of the limitations of short-read sequencing, such as the ability to reliably resolve repeat sequences and large genomic rearrangements. By combining complementary methods with massively parallel DNA sequencing, a greater insight into the biological context of disease mechanisms is now possible. Emerging methodologies, such as advances in nanopore technology, in situ nucleic acid sequencing, and microscopy-based sequencing, will continue the rapid evolution of this area. These new technologies hold many potential applications for hematological disorders, with the promise of precision and personalized medical care in the future.Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

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April 21, 2020

Whole-genome analysis of New Delhi Metallo-Beta-Lactamase-1-producing Acinetobacter haemolyticus from China.

Infections caused by multi-drug resistant Acinetobacter spp. has aroused worldwide attention. With the increasing isolation of non-baumannii Acinetobacter, the nature of infection and resistance associated with them needs to be elaborated. This study aimed to analyze the characteristics of New Delhi Metallo-Beta-Lactamase-1 (NDM-1)-producing Acinetobacter haemolyticus (named sz1652) isolated from Shenzhen city, China.Antibiotic spectrum was analyzed after antimicrobial susceptibility test. Combined disk test (CDT) was used to detecting the metallo-beta-lactamases (MBLs). Transferability of carbapenem resistance was tested by filter mating experiments and plasmid transformation assays. Whole-genome sequencing (WGS) was performed using HiSeq 2000 and PacBio RS system.The A. haemolyticus strain sz1652 was resistant to carbapenems and other tested agents except for amikacin, tigecycline and colistin. The production of MBLs was confirmed by CDT. Transfer of carbapenem resistance was not successful. WGS analysis showed the genome of sz1652 was comprised of chromosome and two plasmids, and sixteen genomic islands (GIs) were predicted. Genes associated with resistance were found in this strain including the beta-lactamase genes blaNDM-1, blaOXA-214 and blaLRA-18, the ?uoroquinolone resistant-related mutations [GyrA subunits (Ser81Ile) and ParC subunits (Ser84Tyr)], and efflux pump genes related to tetracycline and macrolide resistance. Analysis of the genetic environment showed that blaNDM-1was embedded in Tn125 transposon. The Tn125 structure was chromosomally located and shared more than 99% sequence identity with previously reported blaNDM-1 carrying region.The NDM-1-producing A.haemolyticus coexisted multiple durg-resistant determinants. The acquisition of the blaNDM-1 gene was probably facilitated by Tn125 in this strain. Non-A.baumannii species also contain GIs.Copyright © 2019. Published by Elsevier Ltd.

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April 21, 2020

Comparative Genomic Analysis of a Multidrug-Resistant Listeria monocytogenes ST477 Isolate.

Listeria monocytogenes is an opportunistic human foodborne pathogen that causes severe infections with high hospitalization and fatality rates. Clonal complex 9 (CC9) contains a large number of sequence types (STs) and is one of the predominant clones distributed worldwide. However, genetic characteristics of ST477 isolates, which also belong to CC9, have never been examined, and little is known about the detail genomic traits of this food-associated clone. In this study, we sequenced and constructed the whole-genome sequence of an ST477 isolate from a frozen food sample in China and compared it with 58 previously sequenced genomes of 25 human-associated, 5 animal, and 27 food isolates consisting of 6 CC9 and 52 other clones. Phylogenetic analysis revealed that the ST477 clustered with three Canadian ST9 isolates. All phylogeny revealed that CC9 isolates involved in this study consistently possessed the invasion-related gene vip. Mobile genetic elements (MGEs), resistance genes, and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system were elucidated among CC9 isolates. Our ST477 isolate contained a Tn554-like transposon, carrying five arsenical-resistance genes (arsA-arsD, arsR), which was exclusively identified in the CC9 background. Compared with the ST477 genome, three Canadian ST9 isolates shared nonsynonymous nucleotide substitutions in the condensin complex gene smc and cell surface protein genes ftsA and essC. Our findings preliminarily indicate that the extraordinary success of CC9 clone in colonization of different geographical regions is likely due to conserved features harboring MGEs, functional virulence and resistance genes. ST477 and three ST9 genomes are closely related and the distinct differences between them consist primarily of changes in genes involved in multiplication and invasion, which may contribute to the prevalence of ST9 isolates in food and food processing environment.

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April 21, 2020

Detection of transferable oxazolidinone resistance determinants in Enterococcus faecalis and Enterococcus faecium of swine origin in Sichuan Province, China.

The aim of this study was to detect the transferable oxazolidinone resistance determinants (cfr, optrA and poxtA) in E. faecalis and E. faecium of swine origin in Sichuan Province, China.A total of 158 enterococci strains (93 E. faecalis and 65 E. faecium) isolated from 25 large-scale swine farms were screened for the presence of cfr, optrA and poxtA by PCR. The genetic environments of cfr, optrA and poxtA were characterized by whole genome sequencing. Transfer of oxazolidinone resistance determinants was determined by conjugation or electrotransformation experiments.The transferable oxazolidinone resistance determinants, cfr, optrA and poxtA, were detected in zero, six, and one enterococci strains, respectively. The poxtA in one E. faecalis strain was located on a 37,990 bp plasmid, which co-harbored fexB, cat, tet(L) and tet(M), and could be conjugated to E. faecalis JH2-2. One E. faecalis strain harbored two different OptrA variants, including one variant with a single substitution, Q219H, which has not been reported previously. Two optrA-carrying plasmids, pC25-1, with a size of 45,581 bp, and pC54, with a size of 64,500 bp, shared a 40,494 bp identical region that contained genetic context IS1216E-fexA-optrA-erm(A)-IS1216E, which could be electrotransformed into Staphylococcus aureus. Four different chromosomal optrA gene clusters were found in five strains, in which optrA was associated with Tn554 or Tn558 that were inserted into the radC gene.Our study highlights the fact that mobile genetic elements, such as plasmids, IS1216E, Tn554 and Tn558, may facilitate the horizontal transmission of optrA or poxtA.Copyright © 2019. Published by Elsevier Ltd.

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April 21, 2020

Complete genome sequence provides insights into the quorum sensing-related spoilage potential of Shewanella baltica 128 isolated from spoiled shrimp.

Shewanella baltica 128 is a specific spoilage organism (SSO) isolated from the refrigerated shrimp that results in shrimp spoilage. This study reported the complete genome sequencing of this strain, with the primary annotations associated with amino acid transport and metabolism (8.66%), indicating that S. baltica 128 has good potential for degrading proteins. In vitro experiments revealed Shewanella baltica 128 could adapt to the stress conditions by regulating its growth and biofilm formation. Genes that related to the spoilage-related metabolic pathways, including trimethylamine metabolism (torT), sulfur metabolism (cysM), putrescine metabolism (speC), biofilm formation (rpoS) and serine protease production (degS), were identified. Genes (LuxS, pfs, LuxR and qseC) that related to the specific QS system were also identified. Complete genome sequence of S. baltica 128 provide insights into the QS-related spoilage potential, which might provide novel information for the development of new approaches for spoilage detection and prevention based on QS target.Copyright © 2019. Published by Elsevier Inc.

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April 21, 2020

Insect genomes: progress and challenges.

In the wake of constant improvements in sequencing technologies, numerous insect genomes have been sequenced. Currently, 1219 insect genome-sequencing projects have been registered with the National Center for Biotechnology Information, including 401 that have genome assemblies and 155 with an official gene set of annotated protein-coding genes. Comparative genomics analysis showed that the expansion or contraction of gene families was associated with well-studied physiological traits such as immune system, metabolic detoxification, parasitism and polyphagy in insects. Here, we summarize the progress of insect genome sequencing, with an emphasis on how this impacts research on pest control. We begin with a brief introduction to the basic concepts of genome assembly, annotation and metrics for evaluating the quality of draft assemblies. We then provide an overview of genome information for numerous insect species, highlighting examples from prominent model organisms, agricultural pests and disease vectors. We also introduce the major insect genome databases. The increasing availability of insect genomic resources is beneficial for developing alternative pest control methods. However, many opportunities remain for developing data-mining tools that make maximal use of the available insect genome resources. Although rapid progress has been achieved, many challenges remain in the field of insect genomics. © 2019 The Royal Entomological Society.

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April 21, 2020

Complete genome of Pseudomonas sp. DMSP-1 isolated from the Arctic seawater of Kongsfjorden, Svalbard

The genus Pseudomonas is highly metabolically diverse and has colonized a wide range of ecological niches. The strain Pseudomonas sp. DMSP-1 was isolated from Arctic seawater (Kongsfjorden, Svalbard) using dimethylsulfoniopropionate (DMSP) as the sole carbon source. To better understand its role in the Arctic coastal ecosystem, the genome of Pseudomonas sp. strain DMSP-1 was completely sequenced. The genome contained a circular chromosome of 6,282,445?bp with an average GC content of 60.01?mol%. A total of 5510 protein coding genes, 70 tRNA genes and 19 rRNA genes were obtained. However, no genes encoding known enzymes associated with DMSP catabolism were identified in the genome, suggesting that novel DMSP degradation genes might exist in Pseudomonas sp. strain DMSP-1.

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April 21, 2020

The use of Online Tools for Antimicrobial Resistance Prediction by Whole Genome Sequencing in MRSA and VRE.

The antimicrobial resistance (AMR) crisis represents a serious threat to public health and has resulted in concentrated efforts to accelerate development of rapid molecular diagnostics for AMR. In combination with publicly-available web-based AMR databases, whole genome sequencing (WGS) offers the capacity for rapid detection of antibiotic resistance genes. Here we studied the concordance between WGS-based resistance prediction and phenotypic susceptibility testing results for methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus (VRE) clinical isolates using publicly-available tools and databases.Clinical isolates prospectively collected at the University of Pittsburgh Medical Center between December 2016 and December 2017 underwent WGS. Antibiotic resistance gene content was assessed from assembled genomes by BLASTn search of online databases. Concordance between WGS-predicted resistance profile and phenotypic susceptibility as well as sensitivity, specificity, positive and negative predictive values (NPV, PPV) were calculated for each antibiotic/organism combination, using the phenotypic results as the gold standard.Phenotypic susceptibility testing and WGS results were available for 1242 isolate/antibiotic combinations. Overall concordance was 99.3% with a sensitivity, specificity, PPV, NPV of 98.7% (95% CI, 97.2-99.5%), 99.6% (95 % CI, 98.8-99.9%), 99.3% (95% CI, 98.0-99.8%), 99.2% (95% CI, 98.3-99.7%), respectively. Additional identification of point mutations in housekeeping genes increased the concordance to 99.4% and the sensitivity to 99.3% (95% CI, 98.2-99.8%) and NPV to 99.4% (95% CI, 98.4-99.8%).WGS can be used as a reliable predicator of phenotypic resistance for both MRSA and VRE using readily-available online tools.Copyright © 2019. Published by Elsevier Ltd.

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April 21, 2020

Complete genome sequence of Pseudoalteromonas sp. MEBiC 03485, isolated from deep-sea sediment

Pseudoalteromonas strains are widely distributed in the marine environment and most have attracted considerable interest owing to their ability to synthesize biologically active metabolites. In this study, we report and describe the genome sequence of Pseudoalteromonas sp. MEBiC 03485, isolated from the deep-sea sediment of Pacific Ocean at a depth of 2000?m. The complete genome consisted of three contigs with a total genome size of 4,167,407?bp and a GC content of 40.76?l%, and was predicted to contain 4194 protein-coding genes and 131 non-coding RNA genes. The strain MEBiC 03485 genome was also shown to contain genes for diverse metabolic pathways. Genome analysis revealed that the genome of strain MEBiC 03485 was enriched with genes involved in signal transduction, mobile elements, and cold-adaptation, some of which might improve ecological fitness in the deep-sea environment. These findings improve our understanding of microbial adaptation strategies in deep-sea environments.

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April 21, 2020

Extended haplotype phasing of de novo genome assemblies with FALCON-Phase

Haplotype-resolved genome assemblies are important for understanding how combinations of variants impact phenotypes. These assemblies can be created in various ways, such as use of tissues that contain single-haplotype (haploid) genomes, or by co-sequencing of parental genomes, but these approaches can be impractical in many situations. We present FALCON-Phase, which integrates long-read sequencing data and ultra-long-range Hi-C chromatin interaction data of a diploid individual to create high-quality, phased diploid genome assemblies. The method was evaluated by application to three datasets, including human, cattle, and zebra finch, for which high-quality, fully haplotype resolved assemblies were available for benchmarking. Phasing algorithm accuracy was affected by heterozygosity of the individual sequenced, with higher accuracy for cattle and zebra finch (>97%) compared to human (82%). In addition, scaffolding with the same Hi-C chromatin contact data resulted in phased chromosome-scale scaffolds.

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April 21, 2020

Full-length mRNA sequencing and gene expression profiling reveal broad involvement of natural antisense transcript gene pairs in pepper development and response to stresses.

Pepper is an important vegetable with great economic value and unique biological features. In the past few years, significant development has been made towards understanding the huge complex pepper genome; however, pepper functional genomics has not been well studied. To better understand the pepper gene structure and pepper gene regulation, we conducted full-length mRNA sequencing by PacBio sequencing and obtained 57862 high-quality full-length mRNA sequences derived from 18362 previously annotated and 5769 newly detected genes. New gene models were built that combined the full-length mRNA sequences and corrected approximately 500 fragmented gene models from previous annotations. Based on the full-length mRNA, we identified 4114 and 5880 pepper genes forming natural antisense transcript (NAT) genes in-cis and in-trans, respectively. Most of these genes accumulate small RNAs in their overlapping regions. By analyzing these NAT gene expression patterns in our transcriptome data, we identified many NAT pairs responsive to a variety of biological processes in pepper. Pepper formate dehydrogenase 1 (FDH1), which is required for R-gene-mediated disease resistance, may be regulated by nat-siRNAs and participate in a positive feedback loop in salicylic acid biosynthesis during resistance responses. Several cis-NAT pairs and subgroups of trans-NAT genes were responsive to pepper pericarp and placenta development, which may play roles in capsanthin and capsaicin biosynthesis. Using a comparative genomics approach, the evolutionary mechanisms of cis-NATs were investigated, and we found that an increase in intergenic sequences accounted for the loss of most cis-NATs, while transposon insertion contributed to the formation of most new cis-NATs. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

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April 21, 2020

Microbial diversity in the tick Argas japonicus (Acari: Argasidae) with a focus on Rickettsia pathogens.

The soft tick Argas japonicus mainly infests birds and can cause human dermatitis; however, no pathogen has been identified from this tick species in China. In the present study, the microbiota in A. japonicus collected from an epidemic community was explored, and some putative Rickettsia pathogens were further characterized. The results obtained indicated that bacteria in A. japonicus were mainly ascribed to the phyla Proteobacteria, Firmicutes and Actinobacteria. At the genus level, the male A. japonicus harboured more diverse bacteria than the females and nymphs. The bacteria Alcaligenes, Pseudomonas, Rickettsia and Staphylococcus were common in nymphs and adults. The abundance of bacteria belonging to the Rickettsia genus in females and males was 7.27% and 10.42%, respectively. Furthermore, the 16S rRNA gene of Rickettsia was amplified and sequenced, and phylogenetic analysis revealed that 13 sequences were clustered with the spotted fever group rickettsiae (Rickettsia heilongjiangensis and Rickettsia japonica) and three were clustered with Rickettsia limoniae, which suggested that the characterized Rickettsia in A. japonicus were novel putative pathogens and also that the residents were at considerable risk for infection by tick-borne pathogens. © 2019 The Royal Entomological Society.

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April 21, 2020

Pandemic spread of blaKPC-2 among Klebsiella pneumoniae ST11 in China is associated with horizontal transfer mediated by IncFII-like plasmids.

This study aimed to investigate the spread of the blaKPC-2 gene among Klebsiella pneumoniae and to illustrate the mechanism of dissemination of KPC-producing K. pneumoniae (KPC-Kp) ST11 in China.A total of 354 K. pneumoniae isolates were collected from four hospitals in China and were characterized by Multilocus sequence typing (MLST). Mobile genetic elements (MGEs) and pulsed-field gel electrophoresis (PFGE) analysis were used to identify the subtypes of K. pneumoniae ST11. PCR-based amplification and sequencing were performed to analyze Tn1721 transposons and IncFII-like plasmids. Electroporation experiments and whole-genome sequencing (WGS) analysis were used to reveal the genetic environment of the blaKPC-2 gene.As the primary type(87.1%) of KPC-Kp, K. pneumoniae ST11 was not predominant in nonKPC-Kp(3.1%). ST11 KPC-Kp was clonally heterogeneous and could be further classified into eleven MGE types and fourteen PFGE subtypes. Five Tn1721-blaKPC-2 variants were identified on IncFII-like plasmids. The detection rate of IncFII-like plasmids was much higher in ST11 KPC-Kp (100%) compared with non-ST11 KPC-Kp (16.0%) and the nonKPC-Kp group (7.5%). Moreover, the IncFII plasmid (with IIa replicon) was primarily detected on the MGE-F type (61.7%). The IncFIIk plasmid (with IIk replicon) was clustered into two subtypes: MGE-A (28.3%) and -F (41.5%). The detection of the IncFII and IncFIIk plasmids on MGE-A was 57.1% (20/35) and 42.9% (15/35), respectively.We revealed a close correlation between ST11 KPC-Kp and IncFII-like plasmids. Horizontal transfer mediated by IncFII-like plasmids plays an important role in the pandemic expansion of blaKPC-2 among K. pneumoniae ST11 in China. Copyright © 2019. Published by Elsevier B.V.

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