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Asset Tag: Third generation sequencing

April 21, 2020

Extensive intraspecific gene order and gene structural variations in upland cotton cultivars.

Multiple cotton genomes (diploid and tetraploid) have been assembled. However, genomic variations between cultivars of allotetraploid upland cotton (Gossypium hirsutum L.), the most widely planted cotton species in the world, remain unexplored. Here, we use single-molecule long read and Hi-C sequencing technologies to assemble genomes of the two upland cotton cultivars TM-1 and zhongmiansuo24 (ZM24). Comparisons among TM-1 and ZM24 assemblies and the genomes of the diploid ancestors reveal a large amount of genetic variations. Among them, the top three longest structural variations are located on chromosome A08 of the tetraploid upland cotton, which account for ~30% total length of this chromosome. Haplotype analyses of the mapping population derived from these two cultivars and the germplasm panel show suppressed recombination rates in this region. This study provides additional genomic resources for the community, and the identified genetic variations, especially the reduced meiotic recombination on chromosome A08, will help future breeding.

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April 21, 2020

Long-read sequencing unveils IGH-DUX4 translocation into the silenced IGH allele in B-cell acute lymphoblastic leukemia.

IGH@ proto-oncogene translocation is a common oncogenic event in lymphoid lineage cancers such as B-ALL, lymphoma and multiple myeloma. Here, to investigate the interplay between IGH@ proto-oncogene translocation and IGH allelic exclusion, we perform long-read whole-genome and transcriptome sequencing along with epigenetic and 3D genome profiling of Nalm6, an IGH-DUX4 positive B-ALL cell line. We detect significant allelic imbalance on the wild-type over the IGH-DUX4 haplotype in expression and epigenetic data, showing IGH-DUX4 translocation occurs on the silenced IGH allele. In vitro, this reduces the oncogenic stress of DUX4 high-level expression. Moreover, patient samples of IGH-DUX4 B-ALL have similar expression profile and IGH breakpoints as Nalm6, suggesting a common mechanism to allow optimal dosage of non-toxic DUX4 expression.

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April 21, 2020

Urinary tract colonization is enhanced by a plasmid that regulates uropathogenic Acinetobacter baumannii chromosomal genes.

Multidrug resistant (MDR) Acinetobacter baumannii poses a growing threat to global health. Research on Acinetobacter pathogenesis has primarily focused on pneumonia and bloodstream infections, even though one in five A. baumannii strains are isolated from urinary sites. In this study, we highlight the role of A. baumannii as a uropathogen. We develop the first A. baumannii catheter-associated urinary tract infection (CAUTI) murine model using UPAB1, a recent MDR urinary isolate. UPAB1 carries the plasmid pAB5, a member of the family of large conjugative plasmids that represses the type VI secretion system (T6SS) in multiple Acinetobacter strains. pAB5 confers niche specificity, as its carriage improves UPAB1 survival in a CAUTI model and decreases virulence in a pneumonia model. Comparative proteomic and transcriptomic analyses show that pAB5 regulates the expression of multiple chromosomally-encoded virulence factors besides T6SS. Our results demonstrate that plasmids can impact bacterial infections by controlling the expression of chromosomal genes.

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April 21, 2020

Comprehensive identification of the full-length transcripts and alternative splicing related to the secondary metabolism pathways in the tea plant (Camellia sinensis).

Flavonoids, theanine and caffeine are the main secondary metabolites of the tea plant (Camellia sinensis), which account for the tea’s unique flavor quality and health benefits. The biosynthesis pathways of these metabolites have been extensively studied at the transcriptional level, but the regulatory mechanisms are still unclear. In this study, to explore the transcriptome diversity and complexity of tea plant, PacBio Iso-Seq and RNA-seq analysis were combined to obtain full-length transcripts and to profile the changes in gene expression during the leaf development. A total of 1,388,066 reads of insert (ROI) were generated with an average length of 1,762?bp, and more than 54% (755,716) of the ROIs were full-length non-chimeric (FLNC) reads. The Benchmarking Universal Single-Copy Orthologue (BUSCO) completeness was 92.7%. A total of 93,883 non-redundant transcripts were obtained, and 87,395 (93.1%) were new alternatively spliced isoforms. Meanwhile, 7,650 differential expression transcripts (DETs) were identified. A total of 28,980 alternative splicing (AS) events were predicted, including 1,297 differential AS (DAS) events. The transcript isoforms of the key genes involved in the flavonoid, theanine and caffeine biosynthesis pathways were characterized. Additionally, 5,777 fusion transcripts and 9,052 long non-coding RNAs (lncRNAs) were also predicted. Our results revealed that AS potentially plays a crucial role in the regulation of the secondary metabolism of the tea plant. These findings enhanced our understanding of the complexity of the secondary metabolic regulation of tea plants and provided a basis for the subsequent exploration of the regulatory mechanisms of flavonoid, theanine and caffeine biosynthesis in tea plants.

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April 21, 2020

Programmable mutually exclusive alternative splicing for generating RNA and protein diversity.

Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms. We apply this framework to program the DNA-binding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can ultimately be applied to program modular proteins and increase coding capacity of synthetic biological systems.

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April 21, 2020

Genome analysis of the rice coral Montipora capitata.

Corals comprise a biomineralizing cnidarian, dinoflagellate algal symbionts, and associated microbiome of prokaryotes and viruses. Ongoing efforts to conserve coral reefs by identifying the major stress response pathways and thereby laying the foundation to select resistant genotypes rely on a robust genomic foundation. Here we generated and analyzed a high quality long-read based ~886 Mbp nuclear genome assembly and transcriptome data from the dominant rice coral, Montipora capitata from Hawai’i. Our work provides insights into the architecture of coral genomes and shows how they differ in size and gene inventory, putatively due to population size variation. We describe a recent example of foreign gene acquisition via a bacterial gene transfer agent and illustrate the major pathways of stress response that can be used to predict regulatory components of the transcriptional networks in M. capitata. These genomic resources provide insights into the adaptive potential of these sessile, long-lived species in both natural and human influenced environments and facilitate functional and population genomic studies aimed at Hawaiian reef restoration and conservation.

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April 21, 2020

Mutations in sigma 70 transcription factor improves expression of functional eukaryotic membrane proteins in Escherichia coli.

Eukaryotic integral membrane proteins (IMPs) are difficult to study due to low functional expression levels. To investigate factors for efficient biogenesis of eukaryotic IMPs in the prokaryotic model organism Escherichia coli, important, e.g., for isotope-labeling for NMR, we selected for E. coli cells expressing high levels of functional G protein-coupled receptors (GPCRs) by FACS. Utilizing an E. coli strain library with all non-essential genes systematically deleted, we unexpectedly discovered upon whole-genome sequencing that the improved phenotype was not conferred by the deleted genes but by various subtle alterations in the “housekeeping” sigma 70 factor (RpoD). When analyzing effects of the rpoD mutations at the transcriptome level we found that toxic effects incurred on wild-type E. coli during receptor expression were diminished by two independent and synergistic effects: a slower but longer-lasting GPCR biosynthesis and an optimized transcriptional pattern, augmenting growth and expression at low temperature, setting the basis for further bacterial strain engineering.

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April 21, 2020

Identification of a Xist silencing domain by Tiling CRISPR.

Despite essential roles played by long noncoding RNAs (lncRNAs) in development and disease, methods to determine lncRNA cis-elements are lacking. Here, we developed a screening method named “Tiling CRISPR” to identify lncRNA functional domains. Using this approach, we identified Xist A-Repeats as the silencing domain, an observation in agreement with published work, suggesting Tiling CRISPR feasibility. Mechanistic analysis suggested a novel function for Xist A-repeats in promoting Xist transcription. Overall, our method allows mapping of lncRNA functional domains in an unbiased and potentially high-throughput manner to facilitate the understanding of lncRNA functions.

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April 21, 2020

Resistance mechanisms and population structure of highly drug resistant Klebsiella in Pakistan during the introduction of the carbapenemase NDM-1.

Klebsiella pneumoniae is a major threat to public health with the emergence of isolates resistant to most, if not all, useful antibiotics. We present an in-depth analysis of 178 extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae collected from patients resident in a region of Pakistan, during the period 2010-2012, when the now globally-distributed carbapenemase bla-NDM-1 was being acquired by Klebsiella. We observed two dominant lineages, but neither the overall resistance profile nor virulence-associated factors, explain their evolutionary success. Phenotypic analysis of resistance shows few differences between the acquisition of resistance genes and the phenotypic resistance profile, including beta-lactam antibiotics that were used to treat ESBL-positive strains. Resistance against these drugs could be explained by inhibitor-resistant beta-lactamase enzymes, carbapenemases or ampC type beta-lactamases, at least one of which was detected in most, but not all relevant strains analysed. Complete genomes for six selected strains are reported, these provide detailed insights into the mobile elements present in these isolates during the initial spread of NDM-1. The unexplained success of some lineages within this pool of highly resistant strains, and the discontinuity between phenotypic resistance and genotype at the macro level, indicate that intrinsic mechanisms contribute to competitive advantage and/or resistance.

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April 21, 2020

Interspecies conservation of organisation and function between nonhomologous regional centromeres.

Despite the conserved essential function of centromeres, centromeric DNA itself is not conserved. The histone-H3 variant, CENP-A, is the epigenetic mark that specifies centromere identity. Paradoxically, CENP-A normally assembles on particular sequences at specific genomic locations. To gain insight into the specification of complex centromeres, here we take an evolutionary approach, fully assembling genomes and centromeres of related fission yeasts. Centromere domain organization, but not sequence, is conserved between Schizosaccharomyces pombe, S. octosporus and S. cryophilus with a central CENP-ACnp1 domain flanked by heterochromatic outer-repeat regions. Conserved syntenic clusters of tRNA genes and 5S rRNA genes occur across the centromeres of S. octosporus and S. cryophilus, suggesting conserved function. Interestingly, nonhomologous centromere central-core sequences from S. octosporus and S. cryophilus are recognized in S. pombe, resulting in cross-species establishment of CENP-ACnp1 chromatin and functional kinetochores. Therefore, despite the lack of sequence conservation, Schizosaccharomyces centromere DNA possesses intrinsic conserved properties that promote assembly of CENP-A chromatin.

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April 21, 2020

Within-host evolution of Helicobacter pylori shaped by niche-specific adaptation, intragastric migrations and selective sweeps.

The human pathogen Helicobacter pylori displays extensive genetic diversity. While H. pylori is known to evolve during infection, population dynamics inside the gastric environment have not been extensively investigated. Here we obtained gastric biopsies from multiple stomach regions of 16 H. pylori-infected adults, and analyze the genomes of 10 H. pylori isolates from each biopsy. Phylogenetic analyses suggest location-specific evolution and bacterial migration between gastric regions. Migration is significantly more frequent between the corpus and the fundus than with the antrum, suggesting that physiological differences between antral and oxyntic mucosa contribute to spatial partitioning of H. pylori populations. Associations between H. pylori gene polymorphisms and stomach niches suggest that chemotaxis, regulatory functions and outer membrane proteins contribute to specific adaptation to the antral and oxyntic mucosa. Moreover, we show that antibiotics can induce severe population bottlenecks and likely play a role in shaping the population structure of H. pylori.

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April 21, 2020

Genomic features, comparative genomics, and antimicrobial susceptibility patterns of Elizabethkingia bruuniana.

Elizabethkingia bruuniana is a novel species of the Elizabethkingia genus. There is scant information on this microorganism. Here, we report the whole-genome features and antimicrobial susceptibility patterns of E. bruuniana strain EM798-26. Elizabethkingia strain EM798-26 was initially identified as E. miricola. This isolate contained a circular genome of 4,393,011?bp. The whole-genome sequence-based phylogeny revealed that Elizabethkingia strain EM798-26 was in the same group of the type strain E. bruuniana G0146T. Both in silico DNA-DNA hybridization and average nucleotide identity analysis clearly demonstrated that Elizabethkingia strain EM798-26 was a species of E. bruuniana. The pan-genome analysis identified 2,875 gene families in the core genome and 5,199 gene families in the pan genome of eight publicly available E. bruuniana genome sequences. The unique genes accounted for 0.2-12.1% of the pan genome in each E. bruuniana. A total of 59 potential virulence factor homologs were predicted in the whole-genome of E. bruuniana strain EM798-26. This isolate was nonsusceptible to multiple antibiotics, but susceptible to aminoglycosides, minocycline, and levofloxacin. The whole-genome sequence analysis of E. bruuniana EM798-26 revealed 29 homologs of antibiotic resistance-related genes. This study presents the genomic features of E. bruuniana. Knowledge of the genomic characteristics provides valuable insights into a novel species.

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April 21, 2020

Three phylogenetic groups have driven the recent population expansion of Cryptococcus neoformans.

Cryptococcus neoformans (C. neoformans var. grubii) is an environmentally acquired pathogen causing 181,000 HIV-associated deaths each year. We sequenced 699 isolates, primarily C. neoformans from HIV-infected patients, from 5 countries in Asia and Africa. The phylogeny of C. neoformans reveals a recent exponential population expansion, consistent with the increase in the number of susceptible hosts. In our study population, this expansion has been driven by three sub-clades of the C. neoformans VNIa lineage; VNIa-4, VNIa-5 and VNIa-93. These three sub-clades account for 91% of clinical isolates sequenced in our study. Combining the genome data with clinical information, we find that the VNIa-93 sub-clade, the most common sub-clade in Uganda and Malawi, was associated with better outcomes than VNIa-4 and VNIa-5, which predominate in Southeast Asia. This study lays the foundation for further work investigating the dominance of VNIa-4, VNIa-5 and VNIa-93 and the association between lineage and clinical phenotype.

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April 21, 2020

Characterization of Five Escherichia coli Isolates Co-expressing ESBL and MCR-1 Resistance Mechanisms From Different Origins in China

Present study characterized five Escherichia coli co-expressing ESBL and MCR-1 recovered from food, food-producing animals, and companion animals in China. Antimicrobial susceptibility tests, conjugation experiments, and plasmid typing were performed. Whole genome sequencing (WGS) was undertaken for all five isolates using either PacBio RS II or Illumina HiSeq 2500 platforms. The cefotaxime and colistin resistance encoded by blaCTX-M and mcr-1 genes, respectively, was transferable by conjugation either together or separately for all five strains. Interestingly, the ESBL and mcr-1 genes could be co-selected by cefotaxime, while the colistin only selected the mcr-1-carrying plasmids during the conjugation experiments. Five E. coli sequence types (ST88, ST93, ST602, ST162, and ST457) were detected. Although diverse plasmid profiles were identified, IncI2, IncFIB, and IncFII plasmid types were predominant. These five clonally unrelated isolates harbored the mcr-1 gene located on similar plasmid backbones, which showed high nucleotide similarity to plasmid pHNSHP45. The mcr-1 gene can be co-transmitted with blaCTX-Mgenes through IncI2 plasmids with or without ISApl1 in our study. Characterization of these co-existence ESBL and mcr-1 isolates extends our understanding on the dissemination of these resistance markers among bacteria of diverse origins.

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April 21, 2020

Whole-genome-based phylogeny of Bacillus cytotoxicus reveals different clades within the species and provides clues on ecology and evolution.

Bacillus cytotoxicus is a member of the Bacillus cereus group linked to fatal cases of diarrheal disease. Information on B. cytotoxicus is very limited; in particular comprehensive genomic data is lacking. Thus, we applied a genomic approach to characterize B. cytotoxicus and decipher its population structure. To this end, complete genomes of ten B. cytotoxicus were sequenced and compared to the four publicly available full B. cytotoxicus genomes and genomes of other B. cereus group members. Average nucleotide identity, core genome, and pan genome clustering resulted in clear distinction of B. cytotoxicus strains from other strains of the B. cereus group. Genomic content analyses showed that a hydroxyphenylalanine operon is present in B. cytotoxicus, but absent in all other members of the B. cereus group. It enables degradation of aromatic compounds to succinate and pyruvate and was likely acquired from another Bacillus species. It allows for utilization of tyrosine and might have given a B. cytotoxicus ancestor an evolutionary advantage resulting in species differentiation. Plasmid content showed that B. cytotoxicus is flexible in exchanging genes, allowing for quick adaptation to the environment. Genome-based phylogenetic analyses divided the B. cytotoxicus strains into four clades that also differed in virulence gene content.

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