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April 21, 2020  |  

SMRT sequencing analysis reveals the full-length transcripts and alternative splicing patterns in Ananas comosus var. bracteatus.

Ananas comosus var. bracteatus is an herbaceous perennial monocot cultivated as an ornamental plant for its chimeric leaves. Because of its genomic complexity, and because no genomic information is available in the public GenBank database, the complete structure of the mRNA transcript is unclear and there are limited molecular mechanism studies for Ananas comosus var. bracteatus.Three size fractionated full-length cDNA libraries (1-2 kb, 2-3 kb, and 3-6 kb) were constructed and subsequently sequenced in five single-molecule real-time (SMRT) cells (2 cells, 2 cells, and 1 cell, respectively).In total, 19,838 transcripts were identified for alternative splicing (AS) analysis. Among them, 19,185 (96.7%) transcripts were functionally annotated. A total of 9,921 genes were identified by mapping the non-redundant isoforms to the reference genome. A total of 10,649 AS events were identified, the majority of which were intron retention events. The alternatively spliced genes had functions in the basic metabolism processes of the plant such as carbon metabolism, amino acid biosynthesis, and glycolysis. Fourteen genes related to chlorophyll biosynthesis were identified as having AS events. The distribution of the splicing sites and the percentage of conventional and non-canonical AS sites of the genes categorized in pathways related to the albino leaf phenotype (ko00860, ko00195, ko00196, and ko00710) varied greatly. The present results showed that there were 8,316 genes carrying at least one poly (A) site, which generated 21,873 poly (A) sites. These findings indicated that the quality of the gene structure and functional information of the obtained genome was greatly improved, which may facilitate further genetic study of Ananas comosus var. bracteatus.

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April 21, 2020  |  

Rapid antigen diversification through mitotic recombination in the human malaria parasite Plasmodium falciparum.

Malaria parasites possess the remarkable ability to maintain chronic infections that fail to elicit a protective immune response, characteristics that have stymied vaccine development and cause people living in endemic regions to remain at risk of malaria despite previous exposure to the disease. These traits stem from the tremendous antigenic diversity displayed by parasites circulating in the field. For Plasmodium falciparum, the most virulent of the human malaria parasites, this diversity is exemplified by the variant gene family called var, which encodes the major surface antigen displayed on infected red blood cells (RBCs). This gene family exhibits virtually limitless diversity when var gene repertoires from different parasite isolates are compared. Previous studies indicated that this remarkable genome plasticity results from extensive ectopic recombination between var genes during mitotic replication; however, the molecular mechanisms that direct this process to antigen-encoding loci while the rest of the genome remains relatively stable were not determined. Using targeted DNA double-strand breaks (DSBs) and long-read whole-genome sequencing, we show that a single break within an antigen-encoding region of the genome can result in a cascade of recombination events leading to the generation of multiple chimeric var genes, a process that can greatly accelerate the generation of diversity within this family. We also found that recombinations did not occur randomly, but rather high-probability, specific recombination products were observed repeatedly. These results provide a molecular basis for previously described structured rearrangements that drive diversification of this highly polymorphic gene family.

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April 21, 2020  |  

Finding the needle in a haystack: Mapping antifungal drug resistance in fungal pathogen by genomic approaches.

Fungi are ubiquitous on earth and are essential for the maintenance of the global ecological equilibrium. Despite providing benefits to living organisms, they can also target specific hosts and inflict damage. These fungal pathogens are known to affect, for example, plants and mam- mals and thus reduce crop production necessary to sustain food supply and cause mortality in humans and animals. Designing defenses against these fungi is essential for the control of food resources and human health. As far as fungal pathogens are concerned, the principal option has been the use of antifungal agents, also called fungicides when they are used in the environment.

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April 21, 2020  |  

Tools and Strategies for Long-Read Sequencing and De Novo Assembly of Plant Genomes.

The commercial release of third-generation sequencing technologies (TGSTs), giving long and ultra-long sequencing reads, has stimulated the development of new tools for assembling highly contiguous genome sequences with unprecedented accuracy across complex repeat regions. We survey here a wide range of emerging sequencing platforms and analytical tools for de novo assembly, provide background information for each of their steps, and discuss the spectrum of available options. Our decision tree recommends workflows for the generation of a high-quality genome assembly when used in combination with the specific needs and resources of a project.Copyright © 2019 Elsevier Ltd. All rights reserved.

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April 21, 2020  |  

SMRT sequencing revealed the diversity and characteristics of defective interfering RNAs in influenza A (H7N9) virus infection.

Influenza defective interfering (DI) particles are replication-incompetent viruses carrying large internal deletion in the genome. The loss of essential genetic information causes abortive viral replication, which can be rescued by co-infection with a helper virus that possesses an intact genome. Despite reports of DI particles present in seasonal influenza A H1N1 infections, their existence in human infections by the avian influenza A viruses, such as H7N9, has not been studied. Here we report the ubiquitous presence of DI-RNAs in nasopharyngeal aspirates of H7N9-infected patients. Single Molecule Real Time (SMRT) sequencing was first applied and long-read sequencing analysis showed that a variety of H7N9 DI-RNA species were present in the patient samples and human bronchial epithelial cells. In several abundantly expressed DI-RNA species, long overlapping sequences have been identified around at the breakpoint region and the other side of deleted region. Influenza DI-RNA is known as a defective viral RNA with single large internal deletion. Beneficial to the long-read property of SMRT sequencing, double and triple internal deletions were identified in half of the DI-RNA species. In addition, we examined the expression of DI-RNAs in mice infected with sublethal dose of H7N9 virus at different time points. Interestingly, DI-RNAs were abundantly expressed as early as day 2 post-infection. Taken together, we reveal the diversity and characteristics of DI-RNAs found in H7N9-infected patients, cells and animals. Further investigations on this overwhelming generation of DI-RNA may provide important insights into the understanding of H7N9 viral replication and pathogenesis.

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April 21, 2020  |  

Endogenous pararetrovirus sequences are widely present in Citrinae genomes.

Endogenous pararetroviruses (EPRVs) are characterized in several plant genomes and their biological effects have been reported. In this study, hundreds of EPRV segments were identified in six Citrinae genomes. A total of 1034 EPRV segments were identified in the genomes of sweet orange, 2036 in pummelo, 598 in clementine mandarin, 752 in Ichang papeda, 2060 in citron and 245 in atalantia. Genomic analysis indicated that EPRV segments tend to cluster as hot spots in the genomes, particularly on chromosome 2 and 5. Large numbers of simple repeats and transposable elements were identified in the 2-kb flanking regions of the EPRV segments. Comparative genomic analysis and PCR experiments showed that there are highly conserved EPRV segments and species-specific EPRV segments between the Citrinae genomes. Phylogenetic analysis suggested that the integration events of EPRVs could initiate in a common progenitor of Citrinae species and repeatedly occur during the Citrinae divergence.Copyright © 2018 Elsevier B.V. All rights reserved.

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April 21, 2020  |  

Genomic variation and strain-specific functional adaptation in the human gut microbiome during early life.

The human gut microbiome matures towards the adult composition during the first years of life and is implicated in early immune development. Here, we investigate the effects of microbial genomic diversity on gut microbiome development using integrated early childhood data sets collected in the DIABIMMUNE study in Finland, Estonia and Russian Karelia. We show that gut microbial diversity is associated with household location and linear growth of children. Single nucleotide polymorphism- and metagenomic assembly-based strain tracking revealed large and highly dynamic microbial pangenomes, especially in the genus Bacteroides, in which we identified evidence of variability deriving from Bacteroides-targeting bacteriophages. Our analyses revealed functional consequences of strain diversity; only 10% of Finnish infants harboured Bifidobacterium longum subsp. infantis, a subspecies specialized in human milk metabolism, whereas Russian infants commonly maintained a probiotic Bifidobacterium bifidum strain in infancy. Groups of bacteria contributing to diverse, characterized metabolic pathways converged to highly subject-specific configurations over the first two years of life. This longitudinal study extends the current view of early gut microbial community assembly based on strain-level genomic variation.

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April 21, 2020  |  

Whole genome sequencing used in an industrial context reveals a Salmonella laboratory cross-contamination.

In 2013, during a routine laboratory analysis performed on food samples, one finished product from a European factory was tested positive for Salmonella Hadar. At the same period, one environmental isolate in the same laboratory was serotyped Salmonella Hadar. Prior to this event, the laboratory performed a proficiency testing involving a sample spiked with NCTC 9877 Salmonella Hadar. The concomitance of Salmonella Hadar detection led to the suspicion of a laboratory cross-contamination between the Salmonella Hadar isolate used in the laboratory proficiency testing and the Salmonella Hadar isolate found on the finished product by the same laboratory. Since the classical phenotypic serotyping method is able to attribute a serotype to Salmonella isolates with a common antigenic formula, but cannot differentiate strains of the same serotype within the subspecies, whole genome sequencing was used to test the laboratory cross-contamination hypothesis. Additionally, 12 Salmonella Hadar from public databases, available until the time of the event, were included in the whole genome sequencing analysis to better understand the genomic diversity of this serotype in Europe. The outcome of the analysis showed a maximum of ten single nucleotide polymorphisms (SNPs) between the isolates coming from the laboratory and the finished product, and thus confirmed the laboratory cross-contamination. These results combined with all additional investigations done at the factory, allowed to release finished product batches produced and thus circumvented unnecessary food waste and economic losses for the factory. Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.

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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  |  

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  |  

A systematic review of the Trypanosoma cruzi genetic heterogeneity, host immune response and genetic factors as plausible drivers of chronic chagasic cardiomyopathy.

Chagas disease is a complex tropical pathology caused by the kinetoplastid Trypanosoma cruzi. This parasite displays massive genetic diversity and has been classified by international consensus in at least six Discrete Typing Units (DTUs) that are broadly distributed in the American continent. The main clinical manifestation of the disease is the chronic chagasic cardiomyopathy (CCC) that is lethal in the infected individuals. However, one intriguing feature is that only 30-40% of the infected individuals will develop CCC. Some authors have suggested that the immune response, host genetic factors, virulence factors and even the massive genetic heterogeneity of T. cruzi are responsible of this clinical pattern. To date, no conclusive data support the reason why a few percentages of the infected individuals will develop CCC. Therefore, we decided to conduct a systematic review analysing the host genetic factors, immune response, cytokine production, virulence factors and the plausible association of the parasite DTUs and CCC. The epidemiological and clinical implications are herein discussed.

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April 21, 2020  |  

Complete genome sequence unveiled cellulose degradation enzymes and secondary metabolic potentials in Streptomyces sp. CC0208.

Marine Streptomyces sp. CC0208 isolated from the Bohai Bay showed high efficiency of cellulose degradation under optimized fermentation parameters. Also, as one of the bioinformatics-based approaches for the discovery of novel natural product and enzyme effectively, genome mining has been developed and applied widely. Herein, we reported the complete genome sequence of Streptomyces sp. CC0208.Whole-genome sequencing analysis revealed a genome size of 9,325,981?bp with a linear chromosome, GC content of 70.59% and 8487 protein-coding genes. Abundant genes have predicted functions in antibiotic metabolism and enzymes. A 20 enzymes closely associated with cellulose degradation were discovered. A total of 25 biosynthetic gene clusters (BGCs) of secondary metabolites were identified, including diverse classes of natural products. The availability of genome sequence of Streptomyces sp. CC0208 not only will assist in cracking the mechanism of cellulose degradation but also will provide the insights into the significant secondary metabolic potentials for the production of diverse compound classes based on rational strategies. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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April 21, 2020  |  

Alternative polyadenylation coordinates embryonic development, sexual dimorphism and longitudinal growth in Xenopus tropicalis.

RNA alternative polyadenylation contributes to the complexity of information transfer from genome to phenome, thus amplifying gene function. Here, we report the first X. tropicalis resource with 127,914 alternative polyadenylation (APA) sites derived from embryos and adults. Overall, APA networks play central roles in coordinating the maternal-zygotic transition (MZT) in embryos, sexual dimorphism in adults and longitudinal growth from embryos to adults. APA sites coordinate reprogramming in embryos before the MZT, but developmental events after the MZT due to zygotic genome activation. The APA transcriptomes of young adults are more variable than growing adults and male frog APA transcriptomes are more divergent than females. The APA profiles of young females were similar to embryos before the MZT. Enriched pathways in developing embryos were distinct across the MZT and noticeably segregated from adults. Briefly, our results suggest that the minimal functional units in genomes are alternative transcripts as opposed to genes.

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April 21, 2020  |  

Plastid genomes from diverse glaucophyte genera reveal a largely conserved gene content and limited architectural diversity.

Plastid genome (ptDNA) data of Glaucophyta have been limited for many years to the genus Cyanophora. Here, we sequenced the ptDNAs of Gloeochaete wittrockiana, Cyanoptyche gloeocystis, Glaucocystis incrassata, and Glaucocystis sp. BBH. The reported sequences are the first genome-scale plastid data available for these three poorly studied glaucophyte genera. Although the Glaucophyta plastids appear morphologically “ancestral,” they actually bear derived genomes not radically different from those of red algae or viridiplants. The glaucophyte plastid coding capacity is highly conserved (112 genes shared) and the architecture of the plastid chromosomes is relatively simple. Phylogenomic analyses recovered Glaucophyta as the earliest diverging Archaeplastida lineage, but the position of viridiplants as the first branching group was not rejected by the approximately unbiased test. Pairwise distances estimated from 19 different plastid genes revealed that the highest sequence divergence between glaucophyte genera is frequently higher than distances between species of different classes within red algae or viridiplants. Gene synteny and sequence similarity in the ptDNAs of the two Glaucocystis species analyzed is conserved. However, the ptDNA of Gla. incrassata contains a 7.9-kb insertion not detected in Glaucocystis sp. BBH. The insertion contains ten open reading frames that include four coding regions similar to bacterial serine recombinases (two open reading frames), DNA primases, and peptidoglycan aminohydrolases. These three enzymes, often encoded in bacterial plasmids and bacteriophage genomes, are known to participate in the mobilization and replication of DNA mobile elements. It is therefore plausible that the insertion in Gla. incrassata ptDNA is derived from a DNA mobile element.

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April 21, 2020  |  

Long-read sequencing identifies GGC repeat expansions in NOTCH2NLC associated with neuronal intranuclear inclusion disease.

Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disease that is characterized by eosinophilic hyaline intranuclear inclusions in neuronal and somatic cells. The wide range of clinical manifestations in NIID makes ante-mortem diagnosis difficult1-8, but skin biopsy enables its ante-mortem diagnosis9-12. The average onset age is 59.7 years among approximately 140 NIID cases consisting of mostly sporadic and several familial cases. By linkage mapping of a large NIID family with several affected members (Family 1), we identified a 58.1 Mb linked region at 1p22.1-q21.3 with a maximum logarithm of the odds score of 4.21. By long-read sequencing, we identified a GGC repeat expansion in the 5′ region of NOTCH2NLC (Notch 2 N-terminal like C) in all affected family members. Furthermore, we found similar expansions in 8 unrelated families with NIID and 40 sporadic NIID cases. We observed abnormal anti-sense transcripts in fibroblasts specifically from patients but not unaffected individuals. This work shows that repeat expansion in human-specific NOTCH2NLC, a gene that evolved by segmental duplication, causes a human disease.

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