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Asset Tag: Whole Genome Sequencing,

September 21, 2019

Toward complete bacterial genome sequencing through the combined use of multiple next-generation sequencing platforms.

PacBio’s long-read sequencing technologies can be successfully used for a complete bacterial genome assembly using recently developed non-hybrid assemblers in the absence of secondgeneration, high-quality short reads. However, standardized procedures that take into account multiple pre-existing second-generation sequencing platforms are scarce. In addition to Illumina HiSeq and Ion Torrent PGM-based genome sequencing results derived from previous studies, we generated further sequencing data, including from the PacBio RS II platform, and applied various bioinformatics tools to obtain complete genome assemblies for five bacterial strains. Our approach revealed that the hierarchical genome assembly process (HGAP) non-hybrid assembler resulted in nearly complete assemblies at a moderate coverage of ~75x, but that different versions produced non-compatible results requiring post processing. The other two platforms further improved the PacBio assembly through scaffolding and a final error correction.

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September 21, 2019

Functional analysis of the first complete genome sequence of a multidrug resistant sequence type 2 Staphylococcus epidermidis.

Staphylococcus epidermidis is a significant opportunistic pathogen of humans. The ST2 lineage is frequently multidrug resistant and accounts for most of the clinical disease worldwide. However, there are no publically available, closed ST2 genomes and pathogenesis studies have not focused on these strains. We report the complete genome and methylome of BPH0662, a multidrug resistant, hospital adapted, ST2 S. epidermidis, and describe the correlation between resistome and phenotype, as well as demonstrate its relationship to publically available, international ST2 isolates. Furthermore, we delineate the methylome determined by the two type I restriction modification systems present in BPH0662 through heterologous expression in Escherichia coli, allowing the assignment of each system to its corresponding target recognition motif. As the first complete ST2 S. epidermidis genome, BPH0662 provides a valuable reference for future genomic studies of this clinically relevant lineage. Defining the methylome and the construction of these E. coli hosts provides the foundation for the development of molecular tools to bypass restriction modification systems in this lineage that has hitherto proven intractable.

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September 21, 2019

Multiple genome sequences of important beer-spoiling lactic acid bacteria.

Seven strains of important beer-spoiling lactic acid bacteria were sequenced using single-molecule real-time sequencing. Complete genomes were obtained for strains of Lactobacillus paracollinoides, Lactobacillus lindneri, and Pediococcus claussenii The analysis of these genomes emphasizes the role of plasmids as the genomic foundation of beer-spoiling ability. Copyright © 2016 Geissler et al.

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September 21, 2019

in silico Whole Genome Sequencer & Analyzer (iWGS): a computational pipeline to guide the design and analysis of de novo genome sequencing studies.

The availability of genomes across the tree of life is highly biased toward vertebrates, pathogens, human disease models, and organisms with relatively small and simple genomes. Recent progress in genomics has enabled the de novo decoding of the genome of virtually any organism, greatly expanding its potential for understanding the biology and evolution of the full spectrum of biodiversity. The increasing diversity of sequencing technologies, assays, and de novo assembly algorithms have augmented the complexity of de novo genome sequencing projects in non-model organisms. To reduce the costs and challenges in de novo genome sequencing projects and streamline their experimental design and analysis, we developed iWGS (in silico Whole Genome Sequencer and Analyzer), an automated pipeline for guiding the choice of appropriate sequencing strategy and assembly protocols. iWGS seamlessly integrates the four key steps of a de novo genome sequencing project: data generation (through simulation), data quality control, de novo assembly, and assembly evaluation and validation. The last three steps can also be applied to the analysis of real data. iWGS is designed to enable the user to have great flexibility in testing the range of experimental designs available for genome sequencing projects, and supports all major sequencing technologies and popular assembly tools. Three case studies illustrate how iWGS can guide the design of de novo genome sequencing projects and evaluate the performance of a wide variety of user-specified sequencing strategies and assembly protocols on genomes of differing architectures. iWGS, along with a detailed documentation, is freely available at https://github.com/zhouxiaofan1983/iWGS. Copyright © 2016 Author et al.

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September 21, 2019

Recent advances in bioinformatics for fish genomics

In the past few years, we have contributed efforts to ~1/5 of the reported fish genomes. Based on our related experience, here we outline recent advances in bioinformatics for fish genomics, with an emphasis on development of software for genome assembly, genome annotation and evolutionary analysis. This review will be helpful for the new players of genome analysis on both animals and plants. In the past decade, whole genome sequences of approximately 50 fish species have been reported [1]. We have been involved in ~1/5 of these international works from 2014 to 2017, such as mudskippers (2014) [2], Chinese large yellow croaker [3], Chinese barbel fishes [4], Asian arowana [5,6], Channel catfish [7], seahorses [8], Japanese flounder [9], Chinese clearhead icefish [10] and Northern snakehead [11]. We are also in charge of the China Auqatic 10-100-1,000 Genomics Program [12], in which ~100 fish genomes are sequencing targets for the next 3~5 years. Based on our previous experience on fish genomic studies, here we outline recent advances in related bioinformatics for fish genomics to share with public readers. Since the basic informatics includes genome assembly, genome annotation and evolutionary analysis, we discuss them one by one in this order.

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September 21, 2019

Complete genome sequence of the type strain of Macrococcus canis.

The first complete genome sequence of the recently describedMacrococcus canisspecies has been determined for the strain KM45013T(=DSM 101690T= CCOS 969T= CCUG 68920T= CCM 8748T). The strain was isolated from a dog with rhinitis and contains a putative ?-hemolysin and amecB-carrying staphylococcal cassette chromosomemecelement (SCCmecKM45013). Copyright © 2018 Gobeli Brawand et al.

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September 21, 2019

Complete chloroplast genome sequence of the red silk cotton tree (Bombax ceiba)

Bombax ceiba L. is a beautiful and deciduous tree with great ecological and economic importance. The third generation sequencing of chloroplast genome of B. ceiba was conducted on the PacBio sequencing platform (Pacific Biosciences). The complete chloroplast genome was 158,997?bp, which contains a large single-copy (LSC) region (89,021?bp), a small single-copy (SSC) region (21,110?bp), and two inverted repeats (IRs) (24,433?bp). In total, 116 genes were annotated, including 81 protein-coding genes, eight rRNA genes, and 27 tRNA genes. The phylogenetic tree showed that B. ceiba was closely clustered with one clade of Malvaceae.

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September 21, 2019

The complete mitochondrial genome of Bombax ceiba

Bombax ceiba is a beautiful and deciduous tree with important economic and ecological values. Here, we sequenced the intact mitochondrial genome (mitogenome) of B. ceiba on the PacBio sequencing platform (Pacific Biosciences, Menlo Park, CA). The mitogenome is 594,390bp and is comprised of 35 protein-coding genes, two rRNA genes, and 25 tRNA genes. The phylogeny analysis suggested that B. ceiba was closely clustered with the genus Gossypium.

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September 21, 2019

Comparative genomics of enterohemorrhagic Escherichia coli O145:H28 demonstrates a common evolutionary lineage with Escherichia coli O157:H7.

Although serotype O157:H7 is the predominant enterohemorrhagic Escherichia coli (EHEC), outbreaks of non-O157 EHEC that cause severe foodborne illness, including hemolytic uremic syndrome have increased worldwide. In fact, non-O157 serotypes are now estimated to cause over half of all the Shiga toxin-producing Escherichia coli (STEC) cases, and outbreaks of non-O157 EHEC infections are frequently associated with serotypes O26, O45, O103, O111, O121, and O145. Currently, there are no complete genomes for O145 in public databases.We determined the complete genome sequences of two O145 strains (EcO145), one linked to a US lettuce-associated outbreak (RM13514) and one to a Belgium ice-cream-associated outbreak (RM13516). Both strains contain one chromosome and two large plasmids, with genome sizes of 5,737,294 bp for RM13514 and 5,559,008 bp for RM13516. Comparative analysis of the two EcO145 genomes revealed a large core (5,173 genes) and a considerable amount of strain-specific genes. Additionally, the two EcO145 genomes display distinct chromosomal architecture, virulence gene profile, phylogenetic origin of Stx2a prophage, and methylation profile (methylome). Comparative analysis of EcO145 genomes to other completely sequenced STEC and other E. coli and Shigella genomes revealed that, unlike any other known non-O157 EHEC strain, EcO145 ascended from a common lineage with EcO157/EcO55. This evolutionary relationship was further supported by the pangenome analysis of the 10 EHEC str ains. Of the 4,192 EHEC core genes, EcO145 shares more genes with EcO157 than with the any other non-O157 EHEC strains.Our data provide evidence that EcO145 and EcO157 evolved from a common lineage, but ultimately each serotype evolves via a lineage-independent nature to EHEC by acquisition of the core set of EHEC virulence factors, including the genes encoding Shiga toxin and the large virulence plasmid. The large variation between the two EcO145 genomes suggests a distinctive evolutionary path between the two outbreak strains. The distinct methylome between the two EcO145 strains is likely due to the presence of a BsuBI/PstI methyltransferase gene cassette in the Stx2a prophage of the strain RM13514, suggesting a role of horizontal gene transfer-mediated epigenetic alteration in the evolution of individual EHEC strains.

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September 21, 2019

Characterization of multi-drug resistant Enterococcus faecalis isolated from cephalic recording chambers in research macaques (Macaca spp.).

Nonhuman primates are commonly used for cognitive neuroscience research and often surgically implanted with cephalic recording chambers for electrophysiological recording. Aerobic bacterial cultures from 25 macaques identified 72 bacterial isolates, including 15 Enterococcus faecalis isolates. The E. faecalis isolates displayed multi-drug resistant phenotypes, with resistance to ciprofloxacin, enrofloxacin, trimethoprim-sulfamethoxazole, tetracycline, chloramphenicol, bacitracin, and erythromycin, as well as high-level aminoglycoside resistance. Multi-locus sequence typing showed that most belonged to two E. faecalis sequence types (ST): ST 4 and ST 55. The genomes of three representative isolates were sequenced to identify genes encoding antimicrobial resistances and other traits. Antimicrobial resistance genes identified included aac(6′)-aph(2″), aph(3′)-III, str, ant(6)-Ia, tetM, tetS, tetL, ermB, bcrABR, cat, and dfrG, and polymorphisms in parC (S80I) and gyrA (S83I) were observed. These isolates also harbored virulence factors including the cytolysin toxin genes in ST 4 isolates, as well as multiple biofilm-associated genes (esp, agg, ace, SrtA, gelE, ebpABC), hyaluronidases (hylA, hylB), and other survival genes (ElrA, tpx). Crystal violet biofilm assays confirmed that ST 4 isolates produced more biofilm than ST 55 isolates. The abundance of antimicrobial resistance and virulence factor genes in the ST 4 isolates likely relates to the loss of CRISPR-cas. This macaque colony represents a unique model for studying E. faecalis infection associated with indwelling devices, and provides an opportunity to understand the basis of persistence of this pathogen in a healthcare setting.

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September 21, 2019

Whole genome sequence of the soybean aphid, Aphis glycines.

Aphids are emerging as model organisms for both basic and applied research. Of the 5,000 estimated species, only three aphids have published whole genome sequences: the pea aphid Acyrthosiphon pisum, the Russian wheat aphid, Diuraphis noxia, and the green peach aphid, Myzus persicae. We present the whole genome sequence of a fourth aphid, the soybean aphid (Aphis glycines), which is an extreme specialist and an important invasive pest of soybean (Glycine max). The availability of genomic resources is important to establish effective and sustainable pest control, as well as to expand our understanding of aphid evolution. We generated a 302.9 Mbp draft genome assembly for Ap. glycines using a hybrid sequencing approach. This assembly shows high completeness with 19,182 predicted genes, 92% of known Ap. glycines transcripts mapping to contigs, and substantial continuity with a scaffold N50 of 174,505 bp. The assembly represents 95.5% of the predicted genome size of 317.1 Mbp based on flow cytometry. Ap. glycines contains the smallest known aphid genome to date, based on updated genome sizes for 19 aphid species. The repetitive DNA content of the Ap. glycines genome assembly (81.6 Mbp or 26.94% of the 302.9 Mbp assembly) shows a reduction in the number of classified transposable elements compared to Ac. pisum, and likely contributes to the small estimated genome size. We include comparative analyses of gene families related to host-specificity (cytochrome P450’s and effectors), which may be important in Ap. glycines evolution. This Ap. glycines draft genome sequence will provide a resource for the study of aphid genome evolution, their interaction with host plants, and candidate genes for novel insect control methods. Copyright © 2017 Elsevier Ltd. All rights reserved.

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September 21, 2019

Decreased fitness and virulence in ST10 Escherichia coli harboring blaNDM-5 and mcr-1 against a ST4981 strain with blaNDM-5.

Although coexistence of blaNDM-5 and mcr-1 in Escherichia coli has been reported, little is known about the fitness and virulence of such strains. Three carbapenem-resistant Escherichia coli (GZ1, GZ2, and GZ3) successively isolated from one patient in 2015 were investigated for microbiological fitness and virulence. GZ1 and GZ2 were also resistant to colistin. To verify the association between plasmids and fitness, growth kinetics of the transconjugants were performed. We also analyzed genomic sequences of GZ2 and GZ3 using PacBio sequencing. GZ1 and GZ2 (ST10) co-harbored blaNDM-5 and mcr-1, while GZ3 (ST4981) carried only blaNDM-5. GZ3 demonstrated significantly more rapid growth (P < 0.001) and overgrew GZ2 with a competitive index of 1.0157 (4 h) and 2.5207 (24 h). Increased resistance to serum killing and mice mortality was also identified in GZ3. While GZ2 had four plasmids (IncI2, IncX3, IncHI2, IncFII), GZ3 possessed one plasmid (IncFII). The genetic contexts of blaNDM-5 in GZ2 and GZ3 were identical but inserted into different backbones, IncX3 (102,512 bp) and IncFII (91,451 bp), respectively. The growth was not statistically different between the transconjugants with mcr-1 or blaNDM-5 plasmid and recipient (P = 0.6238). Whole genome sequence analysis revealed that 28 virulence genes were specific to GZ3, potentially contributing to increased virulence of GZ3. Decreased fitness and virulence in a mcr-1 and blaNDM-5 co-harboring ST10 E. coli was found alongside a ST4981 strain with only blaNDM-5. Acquisition of mcr-1 or blaNDM-5 plasmid did not lead to considerable fitness costs, indicating the potential for dissemination of mcr-1 and blaNDM-5 in Enterobacteriaceae.

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September 21, 2019

Retrotransposons are the major contributors to the expansion of the Drosophila ananassae Muller F element.

The discordance between genome size and the complexity of eukaryotes can partly be attributed to differences in repeat density. The Muller F element (~5.2 Mb) is the smallest chromosome in Drosophila melanogaster, but it is substantially larger (>18.7 Mb) in D. ananassae To identify the major contributors to the expansion of the F element and to assess their impact, we improved the genome sequence and annotated the genes in a 1.4-Mb region of the D. ananassae F element, and a 1.7-Mb region from the D element for comparison. We find that transposons (particularly LTR and LINE retrotransposons) are major contributors to this expansion (78.6%), while Wolbachia sequences integrated into the D. ananassae genome are minor contributors (0.02%). Both D. melanogaster and D. ananassae F-element genes exhibit distinct characteristics compared to D-element genes (e.g., larger coding spans, larger introns, more coding exons, and lower codon bias), but these differences are exaggerated in D. ananassae Compared to D. melanogaster, the codon bias observed in D. ananassae F-element genes can primarily be attributed to mutational biases instead of selection. The 5′ ends of F-element genes in both species are enriched in dimethylation of lysine 4 on histone 3 (H3K4me2), while the coding spans are enriched in H3K9me2. Despite differences in repeat density and gene characteristics, D. ananassae F-element genes show a similar range of expression levels compared to genes in euchromatic domains. This study improves our understanding of how transposons can affect genome size and how genes can function within highly repetitive domains. Copyright © 2017 Leung et al.

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September 21, 2019

A distinct and genetically diverse lineage of the hybrid fungal pathogen Verticillium longisporum population causes stem striping in British oilseed rape.

Population genetic structures illustrate evolutionary trajectories of organisms adapting to differential environmental conditions. Verticillium stem striping disease on oilseed rape was mainly observed in continental Europe, but has recently emerged in the United Kingdom. The disease is caused by the hybrid fungal species Verticillium longisporum that originates from at least three separate hybridization events, yet hybrids between Verticillium progenitor species A1 and D1 are mainly responsible for Verticillium stem striping. We reveal a hitherto un-described dichotomy within V. longisporum lineage A1/D1 that correlates with the geographic distribution of the isolates with an ‘A1/D1 West’ and an ‘A1/D1 East’ cluster. Genome comparison between representatives of the A1/D1 West and East clusters excluded population distinctiveness through separate hybridization events. Remarkably, the A1/D1 West population that is genetically more diverse than the entire A1/D1 East cluster caused the sudden emergence of Verticillium stem striping in the UK, whereas in continental Europe Verticillium stem striping is predominantly caused by the more genetically uniform A1/D1 East population. The observed genetic diversity of the A1/D1 West population argues against a recent introduction of the pathogen into the UK, but rather suggests that the pathogen previously established in the UK and remained latent or unnoticed as oilseed rape pathogen until recently.© 2017 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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