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April 21, 2020

Bioinformatic analysis of the complete genome sequence of Pectobacterium carotovorum subsp. brasiliense BZA12 and candidate effector screening

AbstractPectobacterium carotovorum subsp. brasiliense (Pcb) is a gram-negative, plant pathogenic bacterium of the soft rot Enterobacteriaceae (SRE) family. We present the complete genome sequence of Pcb strain BZA12, which reveals that Pcb strain BZA12 carries a single 4,924,809 bp chromosome with 51.97% GC content and comprises 4508 predicted protein-coding genes.Geneannotationofthese genes utilizedGO, KEGG,and COG databases.Incomparison withthree closely related soft-rot pathogens, strain BZA12 has 3797 gene families, among which 3107 gene families are identified as orthologous with those of both P. carotovorum subsp. carotovorum PCC21 and P. carotovorum subsp. odoriferum BCS7, as well as 36 putative Unique Gene Families. We selected five putative effectors from the BZA12 genome and transiently expressed them in Nicotiana benthamiana. Candidate effector A12GL002483 was localized in the cell nucleus and induced cell death. This study provides a foundation for a better understanding of the genomic structure and function of Pcb, particularly in the discovery of potential pathogenic factors and for the development of more effective strategies against this pathogen.

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April 21, 2020

Investigating the bacterial microbiota of traditional fermented dairy products using propidium monoazide with single-molecule real-time sequencing.

Traditional fermented dairy foods have been the major components of the Mongolian diet for millennia. In this study, we used propidium monoazide (PMA; binds to DNA of nonviable cells so that only viable cells are enumerated) and single-molecule real-time sequencing (SMRT) technology to investigate the total and viable bacterial compositions of 19 traditional fermented dairy foods, including koumiss from Inner Mongolia (KIM), koumiss from Mongolia (KM), and fermented cow milk from Mongolia (CM); sample groups treated with PMA were designated PKIM, PKM, and PCM. Full-length 16S rRNA sequencing identified 195 bacterial species in 121 genera and 13 phyla in PMA-treated and untreated samples. The PMA-treated and untreated samples differed significantly in their bacterial community composition and a-diversity values. The predominant species in KM, KIM, and CM were Lactobacillus helveticus, Streptococcus parauberis, and Lactobacillus delbrueckii, whereas the predominant species in PKM, PKIM, and PCM were Enterobacter xiangfangensis, Lactobacillus helveticus, and E. xiangfangensis, respectively. Weighted and unweighted principal coordinate analyses showed a clear clustering pattern with good separation and only minor overlapping. In addition, a pure culture method was performed to obtain lactic acid bacteria resources in dairy samples according to the results of SMRT sequencing. A total of 102 LAB strains were identified and Lb. helveticus (68.63%) was the most abundant, in agreement with SMRT sequencing results. Our results revealed that the bacterial communities of traditional dairy foods are complex and vary by type of fermented dairy product. The PMA treatment induced significant changes in bacterial community structure.Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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April 21, 2020

Novel trimethoprim resistance gene dfrA34 identified in Salmonella Heidelberg in the USA.

Trimethoprim/sulfamethoxazole is a synthetic antibiotic combination recommended for the treatment of complicated non-typhoidal Salmonella infections in humans. Resistance to trimethoprim/sulfamethoxazole is mediated by the acquisition of mobile genes, requiring both a dfr gene (trimethoprim resistance) and a sul gene (sulfamethoxazole resistance) for a clinical resistance phenotype (MIC =4/76?mg/L). In 2017, the CDC investigated a multistate outbreak caused by a Salmonella enterica serotype Heidelberg strain with trimethoprim/sulfamethoxazole resistance, in which sul genes but no known dfr genes were detected.To characterize and describe the molecular mechanism of trimethoprim resistance in a Salmonella Heidelberg outbreak isolate.Illumina sequencing data for one outbreak isolate revealed a 588?bp ORF encoding a putative dfr gene. This gene was cloned into Escherichia coli and resistance to trimethoprim was measured by broth dilution and Etest. Phylogenetic analysis of previously reported dfrA genes was performed using MEGA. Long-read sequencing was conducted to determine the context of the novel dfr gene.The novel dfr gene, named dfrA34, conferred trimethoprim resistance (MIC =32?mg/L) when cloned into E. coli. Based on predicted amino acid sequences, dfrA34 shares less than 50% identity with other known dfrA genes. The dfrA34 gene is located in a class 1 integron in a multiresistance region of an IncC plasmid, adjacent to a sul gene, thus conferring clinical trimethoprim/sulfamethoxazole resistance. Additionally, dfrA34 is associated with ISCR1, enabling easy transmission between other plasmids and bacterial strains.

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April 21, 2020

Genome-Scale Sequence Disruption Following Biolistic Transformation in Rice and Maize.

Biolistic transformation delivers nucleic acids into plant cells by bombarding the cells with microprojectiles, which are micron-scale, typically gold particles. Despite the wide use of this technique, little is known about its effect on the cell’s genome. We biolistically transformed linear 48-kb phage lambda and two different circular plasmids into rice (Oryza sativa) and maize (Zea mays) and analyzed the results by whole genome sequencing and optical mapping. Although some transgenic events showed simple insertions, others showed extreme genome damage in the form of chromosome truncations, large deletions, partial trisomy, and evidence of chromothripsis and breakage-fusion bridge cycling. Several transgenic events contained megabase-scale arrays of introduced DNA mixed with genomic fragments assembled by nonhomologous or microhomology-mediated joining. Damaged regions of the genome, assayed by the presence of small fragments displaced elsewhere, were often repaired without a trace, presumably by homology-dependent repair (HDR). The results suggest a model whereby successful biolistic transformation relies on a combination of end joining to insert foreign DNA and HDR to repair collateral damage caused by the microprojectiles. The differing levels of genome damage observed among transgenic events may reflect the stage of the cell cycle and the availability of templates for HDR. © 2019 American Society of Plant Biologists. All rights reserved.

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April 21, 2020

Fast and accurate genomic analyses using genome graphs.

The human reference genome serves as the foundation for genomics by providing a scaffold for alignment of sequencing reads, but currently only reflects a single consensus haplotype, thus impairing analysis accuracy. Here we present a graph reference genome implementation that enables read alignment across 2,800 diploid genomes encompassing 12.6 million SNPs and 4.0 million insertions and deletions (indels). The pipeline processes one whole-genome sequencing sample in 6.5?h using a system with 36?CPU cores. We show that using a graph genome reference improves read mapping sensitivity and produces a 0.5% increase in variant calling recall, with unaffected specificity. Structural variations incorporated into a graph genome can be genotyped accurately under a unified framework. Finally, we show that iterative augmentation of graph genomes yields incremental gains in variant calling accuracy. Our implementation is an important advance toward fulfilling the promise of graph genomes to radically enhance the scalability and accuracy of genomic analyses.

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April 21, 2020

Full-length transcriptome analysis of Litopenaeus vannamei reveals transcript variants involved in the innate immune system.

To better understand the immune system of shrimp, this study combined PacBio isoform sequencing (Iso-Seq) and Illumina paired-end short reads sequencing methods to discover full-length immune-related molecules of the Pacific white shrimp, Litopenaeus vannamei. A total of 72,648 nonredundant full-length transcripts (unigenes) were generated with an average length of 2545 bp from five main tissues, including the hepatopancreas, cardiac stomach, heart, muscle, and pyloric stomach. These unigenes exhibited a high annotation rate (62,164, 85.57%) when compared against NR, NT, Swiss-Prot, Pfam, GO, KEGG and COG databases. A total of 7544 putative long noncoding RNAs (lncRNAs) were detected and 1164 nonredundant full-length transcripts (449 UniTransModels) participated in the alternative splicing (AS) events. Importantly, a total of 5279 nonredundant full-length unigenes were successfully identified, which were involved in the innate immune system, including 9 immune-related processes, 19 immune-related pathways and 10 other immune-related systems. We also found wide transcript variants, which increased the number and function complexity of immune molecules; for example, toll-like receptors (TLRs) and interferon regulatory factors (IRFs). The 480 differentially expressed genes (DEGs) were significantly higher or tissue-specific expression patterns in the hepatopancreas compared with that in other four tested tissues (FDR <0.05). Furthermore, the expression levels of six selected immune-related DEGs and putative IRFs were validated using real-time PCR technology, substantiating the reliability of the PacBio Iso-seq results. In conclusion, our results provide new genetic resources of long-read full-length transcripts data and information for identifying immune-related genes, which are an invaluable transcriptomic resource as genomic reference, especially for further exploration of the innate immune and defense mechanisms of shrimp. Copyright © 2019 Elsevier Ltd. All rights reserved.

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April 21, 2020

The interplay between microRNA and alternative splicing of linear and circular RNAs in eleven plant species.

MicroRNA (miRNA) and alternative splicing (AS)-mediated post-transcriptional regulation has been extensively studied in most eukaryotes. However, the interplay between AS and miRNAs has not been explored in plants. To our knowledge, the overall profile of miRNA target sites in circular RNAs (circRNA) generated by alternative back splicing has never been reported previously. To address the challenge, we identified miRNA target sites located in alternatively spliced regions of the linear and circular splice isoforms using the up-to-date single-molecule real-time (SMRT) isoform sequencing (Iso-Seq) and Illumina sequencing data in eleven plant species.In total, we identified 399?401 and 114?574 AS events from linear and circular RNAs, respectively. Among them, there were 64?781 and 41?146 miRNA target sites located in linear and circular AS region, respectively. In addition, we found 38?913 circRNAs to be overlapping with 45?648 AS events of its own parent isoforms, suggesting circRNA regulation of AS of linear RNAs by forming R-loop with the genomic locus. Here, we present a comprehensive database of miRNA targets in alternatively spliced linear and circRNAs (ASmiR) and a web server for deposition and identification of miRNA target sites located in the alternatively spliced region of linear and circular RNAs. This database is accompanied by an easy-to-use web query interface for meaningful downstream analysis. Plant research community can submit user-defined datasets to the web service to search AS regions harboring small RNA target sites. In conclusion, this study provides an unprecedented resource to understand regulatory relationships between miRNAs and AS in both gymnosperms and angiosperms.The readily accessible database and web-based tools are available at http://forestry.fafu.edu.cn/bioinfor/db/ASmiR.Supplementary data are available at Bioinformatics online. © The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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April 21, 2020

Hybrid sequencing-based personal full-length transcriptomic analysis implicates proteostatic stress in metastatic ovarian cancer.

Comprehensive molecular characterization of myriad somatic alterations and aberrant gene expressions at personal level is key to precision cancer therapy, yet limited by current short-read sequencing technology, individualized catalog of complete genomic and transcriptomic features is thus far elusive. Here, we integrated second- and third-generation sequencing platforms to generate a multidimensional dataset on a patient affected by metastatic epithelial ovarian cancer. Whole-genome and hybrid transcriptome dissection captured global genetic and transcriptional variants at previously unparalleled resolution. Particularly, single-molecule mRNA sequencing identified a vast array of unannotated transcripts, novel long noncoding RNAs and gene chimeras, permitting accurate determination of transcription start, splice, polyadenylation and fusion sites. Phylogenetic and enrichment inference of isoform-level measurements implicated early functional divergence and cytosolic proteostatic stress in shaping ovarian tumorigenesis. A complementary imaging-based high-throughput drug screen was performed and subsequently validated, which consistently pinpointed proteasome inhibitors as an effective therapeutic regime by inducing protein aggregates in ovarian cancer cells. Therefore, our study suggests that clinical application of the emerging long-read full-length analysis for improving molecular diagnostics is feasible and informative. An in-depth understanding of the tumor transcriptome complexity allowed by leveraging the hybrid sequencing approach lays the basis to reveal novel and valid therapeutic vulnerabilities in advanced ovarian malignancies.

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April 21, 2020

Retrospective whole-genome sequencing analysis distinguished PFGE and drug-resistance-matched retail meat and clinical Salmonella isolates.

Non-typhoidal Salmonella is a leading cause of outbreak and sporadic-associated foodborne illnesses in the United States. These infections have been associated with a range of foods, including retail meats. Traditionally, pulsed-field gel electrophoresis (PFGE) and antibiotic susceptibility testing (AST) have been used to facilitate public health investigations of Salmonella infections. However, whole-genome sequencing (WGS) has emerged as an alternative tool that can be routinely implemented. To assess its potential in enhancing integrated surveillance in Pennsylvania, USA, WGS was used to directly compare the genetic characteristics of 7 retail meat and 43 clinical historic Salmonella isolates, subdivided into 3 subsets based on PFGE and AST results, to retrospectively resolve their genetic relatedness and identify antimicrobial resistance (AMR) determinants. Single nucleotide polymorphism (SNP) analyses revealed that the retail meat isolates within S. Heidelberg, S. Typhimurium var. O5- subset 1 and S. Typhimurium var. O5- subset 2 were separated from each primary PFGE pattern-matched clinical isolate by 6-12, 41-96 and 21-81 SNPs, respectively. Fifteen resistance genes were identified across all isolates, including fosA7, a gene only recently found in a limited number of Salmonella and a =95?%?phenotype to genotype correlation was observed for all tested antimicrobials. Moreover, AMR was primarily plasmid-mediated in S. Heidelberg and S. Typhimurium var. O5- subset 2, whereas AMR was chromosomally carried in S. Typhimurium var. O5- subset 1. Similar plasmids were identified in both the retail meat and clinical isolates. Collectively, these data highlight the utility of WGS in retrospective analyses and enhancing integrated surveillance for Salmonella from multiple sources.

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April 21, 2020

Long-read sequence capture of the haemoglobin gene clusters across codfish species.

Combining high-throughput sequencing with targeted sequence capture has become an attractive tool to study specific genomic regions of interest. Most studies have so far focused on the exome using short-read technology. These approaches are not designed to capture intergenic regions needed to reconstruct genomic organization, including regulatory regions and gene synteny. Here, we demonstrate the power of combining targeted sequence capture with long-read sequencing technology for comparative genomic analyses of the haemoglobin (Hb) gene clusters across eight species separated by up to 70 million years. Guided by the reference genome assembly of the Atlantic cod (Gadus morhua) together with genome information from draft assemblies of selected codfishes, we designed probes covering the two Hb gene clusters. Use of custom-made barcodes combined with PacBio RSII sequencing led to highly continuous assemblies of the LA (~100 kb) and MN (~200 kb) clusters, which include syntenic regions of coding and intergenic sequences. Our results revealed an overall conserved genomic organization of the Hb genes within this lineage, yet with several, lineage-specific gene duplications. Moreover, for some of the species examined, we identified amino acid substitutions at two sites in the Hbb1 gene as well as length polymorphisms in its regulatory region, which has previously been linked to temperature adaptation in Atlantic cod populations. This study highlights the use of targeted long-read capture as a versatile approach for comparative genomic studies by generation of a cross-species genomic resource elucidating the evolutionary history of the Hb gene family across the highly divergent group of codfishes. © 2018 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

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April 21, 2020

Population Genome Sequencing of the Scab Fungal Species Venturia inaequalis, Venturia pirina, Venturia aucupariae and Venturia asperata.

The Venturia genus comprises fungal species that are pathogens on Rosaceae host plants, including V. inaequalis and V. asperata on apple, V. aucupariae on sorbus and V. pirina on pear. Although the genetic structure of V. inaequalis populations has been investigated in detail, genomic features underlying these subdivisions remain poorly understood. Here, we report whole genome sequencing of 87 Venturia strains that represent each species and each population within V. inaequalis We present a PacBio genome assembly for the V. inaequalis EU-B04 reference isolate. The size of selected genomes was determined by flow cytometry, and varied from 45 to 93 Mb. Genome assemblies of V. inaequalis and V. aucupariae contain a high content of transposable elements (TEs), most of which belong to the Gypsy or Copia LTR superfamilies and have been inactivated by Repeat-Induced Point mutations. The reference assembly of V. inaequalis presents a mosaic structure of GC-equilibrated regions that mainly contain predicted genes and AT-rich regions, mainly composed of TEs. Six pairs of strains were identified as clones. Single-Nucleotide Polymorphism (SNP) analysis between these clones revealed a high number of SNPs that are mostly located in AT-rich regions due to misalignments and allowed determining a false discovery rate. The availability of these genome sequences is expected to stimulate genetics and population genomics research of Venturia pathogens. Especially, it will help understanding the evolutionary history of Venturia species that are pathogenic on different hosts, a history that has probably been substantially influenced by TEs.Copyright © 2019 Le Cam et al.

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April 21, 2020

High Quality Draft Genome of Arogyapacha (Trichopus zeylanicus), an Important Medicinal Plant Endemic to Western Ghats of India.

Arogyapacha, the local name of Trichopus zeylanicus, is a rare, indigenous medicinal plant of India. This plant is famous for its traditional use as an instant energy stimulant. So far, no genomic resource is available for this important plant and hence its metabolic pathways are poorly understood. Here, we report on a high-quality draft assembly of approximately 713.4 Mb genome of T. zeylanicus, first draft genome from the genus Trichopus The assembly was generated in a hybrid approach using Illumina short-reads and Pacbio longer-reads. The total assembly comprised of 22601 scaffolds with an N50 value of 433.3 Kb. We predicted 34452 protein coding genes in T. zeylanicus genome and found that a significant portion of these predicted genes were associated with various secondary metabolite biosynthetic pathways. Comparative genome analysis revealed extensive gene collinearity between T. zeylanicus and its closely related plant species. The present genome and annotation data provide an essential resource to speed-up the research on secondary metabolism, breeding and molecular evolution of T. zeylanicus. Copyright © 2019 Chellappan et al.

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April 21, 2020

Reference genome sequences of two cultivated allotetraploid cottons, Gossypium hirsutum and Gossypium barbadense.

Allotetraploid cotton species (Gossypium hirsutum and Gossypium barbadense) have long been cultivated worldwide for natural renewable textile fibers. The draft genome sequences of both species are available but they are highly fragmented and incomplete1-4. Here we report reference-grade genome assemblies and annotations for G. hirsutum accession Texas Marker-1 (TM-1) and G. barbadense accession 3-79 by integrating single-molecule real-time sequencing, BioNano optical mapping and high-throughput chromosome conformation capture techniques. Compared with previous assembled draft genomes1,3, these genome sequences show considerable improvements in contiguity and completeness for regions with high content of repeats such as centromeres. Comparative genomics analyses identify extensive structural variations that probably occurred after polyploidization, highlighted by large paracentric/pericentric inversions in 14 chromosomes. We constructed an introgression line population to introduce favorable chromosome segments from G. barbadense to G. hirsutum, allowing us to identify 13 quantitative trait loci associated with superior fiber quality. These resources will accelerate evolutionary and functional genomic studies in cotton and inform future breeding programs for fiber improvement.

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April 21, 2020

Genomic analysis of three Clostridioides difficile isolates from urban water sources.

We investigated inflow of a wastewater treatment plant and sediment of an urban lake for the presence of Clostridioides difficile by cultivation and PCR. Among seven colonies we sequenced the complete genomes of three: two non-toxigenic isolates from wastewater and one toxigenic isolate from the urban lake. For all obtained isolates, a close genomic relationship with human-derived isolates was observed.Copyright © 2019 Elsevier Ltd. All rights reserved.

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April 21, 2020

The developmental dynamics of the Populus stem transcriptome.

The Populus shoot undergoes primary growth (longitudinal growth) followed by secondary growth (radial growth), which produces biomass that is an important source of energy worldwide. We adopted joint PacBio Iso-Seq and RNA-seq analysis to identify differentially expressed transcripts along a developmental gradient from the shoot apex to the fifth internode of Populus Nanlin895. We obtained 87 150 full-length transcripts, including 2081 new isoforms and 62 058 new alternatively spliced isoforms, most of which were produced by intron retention, that were used to update the Populus annotation. Among these novel isoforms, there are 1187 long non-coding RNAs and 356 fusion genes. Using this annotation, we found 15 838 differentially expressed transcripts along the shoot developmental gradient, of which 1216 were transcription factors (TFs). Only a few of these genes were reported previously. The differential expression of these TFs suggests that they may play important roles in primary and secondary growth. AP2, ARF, YABBY and GRF TFs are highly expressed in the apex, whereas NAC, bZIP, PLATZ and HSF TFs are likely to be important for secondary growth. Overall, our findings provide evidence that long-read sequencing can complement short-read sequencing for cataloguing and quantifying eukaryotic transcripts and increase our understanding of the vital and dynamic process of shoot development. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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