Closing Clostridium botulinum Group III Genomes Using Long-Read Sequencing.
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ABSTRACT:
SUBMITTER: Woudstra C
PROVIDER: S-EPMC8354527 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Clostridium botulinum is a Gram-positive, spore-forming anaerobic bacterium that produces botulinum neurotoxin (BoNT). Closing their genomes provides information about their neurotoxin clusters' arrangement(s) and their location (e.g., chromosome or plasmid) which cannot be assessed using draft genomes. Therefore, we tested the use of long-read sequencing (nanopore sequencing) in combination with short-read sequencing to close two toxin-producing strains. These genomes could be used by the Public Health Emergency Preparedness and Response staff during botulism outbreaks. The genomes of two toxin-producing C. botulinum strains, one from an environmental sample (83F_CFSAN034202) and the other from a clinical sample (CDC51232_CFSAN034200) were sequenced using MinION and MiSeq devices. The genomes, including the chromosomes and the plasmids, were closed by a combination of long-read and short-read sequencing. They belonged to different C. botulinum sequence types (STs), with 83F belonging to ST4 and CDC51232 to ST7. A whole genome single nucleotide polymorphism (SNP) analysis clustered these two strains with strains in lineage 2 (e.g., 6CDC297) and 4 (e.g., NCTC2916) from Group I, respectively. These two strains were also bivalent strains with the BoNTB and BoNTA4 clusters located in the larger plasmid for CDC51232, and the BoNTB and BoNTA1 clusters located both in the chromosome for 83F. Overall, this study showed the advantage of combining these two sequencing methods to obtain high quality closed C. botulinum genomes that could be used for SNP phylogenies (source tracking) as well as for fast identification of BoNT clusters and their gene arrangements.
| S-EPMC7050642 | biostudies-literature
Project description:Recent developments in whole genome sequencing have made a substantial contribution to understanding the genomes, neurotoxins and biology of Clostridium botulinum Group I (proteolytic C. botulinum) and C. botulinum Group II (non-proteolytic C. botulinum). Two different approaches are used to study genomics in these bacteria; comparative whole genome microarrays and direct comparison of complete genome DNA sequences. The properties of the different types of neurotoxin formed, and different neurotoxin gene clusters found in C. botulinum Groups I and II are explored. Specific examples of botulinum neurotoxin genes are chosen for an in-depth discussion of neurotoxin gene evolution. The most recent cases of foodborne botulism are summarised.
| S-EPMC4430135 | biostudies-literature
Project description:Obtaining high-quality sequence continuity of complex regions of recent segmental duplication remains one of the major challenges of finishing genome assemblies. In the human and mouse genomes, this was achieved by targeting large-insert clones using costly and laborious capillary-based sequencing approaches. Sanger shotgun sequencing of clone inserts, however, has now been largely abandoned, leaving most of these regions unresolved in newer genome assemblies generated primarily by next-generation sequencing hybrid approaches. Here we show that it is possible to resolve regions that are complex in a genome-wide context but simple in isolation for a fraction of the time and cost of traditional methods using long-read single molecule, real-time (SMRT) sequencing and assembly technology from Pacific Biosciences (PacBio). We sequenced and assembled BAC clones corresponding to a 1.3-Mbp complex region of chromosome 17q21.31, demonstrating 99.994% identity to Sanger assemblies of the same clones. We targeted 44 differences using Illumina sequencing and find that PacBio and Sanger assemblies share a comparable number of validated variants, albeit with different sequence context biases. Finally, we targeted a poorly assembled 766-kbp duplicated region of the chimpanzee genome and resolved the structure and organization for a fraction of the cost and time of traditional finishing approaches. Our data suggest a straightforward path for upgrading genomes to a higher quality finished state.
| S-EPMC3975067 | biostudies-literature
Project description:In animals, botulism is commonly sustained by botulinum neurotoxin C, D or their mosaic variants, which are produced by anaerobic bacteria included in Clostridium botulinum group III. In this study, a WGS has been applied to a large collection of C. botulinum group III field strains in order to expand the knowledge on these BoNT-producing Clostridia and to evaluate the potentiality of this method for epidemiological investigations. Sixty field strains were submitted to WGS, and the results were analyzed with respect to epidemiological information and compared to published sequences. The strains were isolated from biological or environmental samples collected in animal botulism outbreaks which occurred in Italy from 2007 to 2016. The new sequenced strains belonged to subspecific groups, some of which were already defined, while others were newly characterized, peculiar to Italian strains and contained genomic features not yet observed. This included, in particular, two new flicC types (VI and VII) and new plasmids which widen the known plasmidome of the species. The extensive genome exploration shown in this study improves the C. botulinum and related species classification scheme, enriching it with new strains of rare genotypes and permitting the highest grade of discrimination among strains for forensic and epidemiological applications.
| S-EPMC8624178 | biostudies-literature
Project description:BackgroundThe marine diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum are valuable model organisms for exploring the evolution, diversity and ecology of this important algal group. Their reference genomes, published in 2004 and 2008, respectively, were the product of traditional Sanger sequencing. In the case of T. pseudonana, optical restriction site mapping was employed to further clarify and contextualize chromosome-level scaffolds. While both genomes are considered highly accurate and reasonably contiguous, they still contain many unresolved regions and unordered/unlinked scaffolds.ResultsWe have used Oxford Nanopore Technologies long-read sequencing to update and validate the quality and contiguity of the T. pseudonana and P. tricornutum genomes. Fine-scale assessment of our long-read derived genome assemblies allowed us to resolve previously uncertain genomic regions, further characterize complex structural variation, and re-evaluate the repetitive DNA content of both genomes. We also identified 1862 previously undescribed genes in T. pseudonana. In P. tricornutum, we used transposable element detection software to identify 33 novel copia-type LTR-RT insertions, indicating ongoing activity and rapid expansion of this superfamily as the organism continues to be maintained in culture. Finally, Bionano optical mapping of P. tricornutum chromosomes was combined with long-read sequence data to explore the potential of long-read sequencing and optical mapping for resolving haplotypes.ConclusionDespite its potential to yield highly contiguous scaffolds, long-read sequencing is not a panacea. Even for relatively small nuclear genomes such as those investigated herein, repetitive DNA sequences cause problems for current genome assembly algorithms. Determining whether a long-read derived genomic assembly is 'better' than one produced using traditional sequence data is not straightforward. Our revised reference genomes for P. tricornutum and T. pseudonana nevertheless provide additional insight into the structure and evolution of both genomes, thereby providing a more robust foundation for future diatom research.
| S-EPMC8147415 | biostudies-literature
Project description:BackgroundClostridium botulinum strains can be divided into four physiological groups that are sufficiently diverged to be considered as separate species. Here we present the first complete genome of a C. botulinum strain from physiological group III, causing animal botulism. We also compare the sequence to three new draft genomes from the same physiological group.ResultsThe 2.77 Mb chromosome was highly conserved between the isolates and also closely related to that of C. novyi. However, the sequence was very different from the human C. botulinum group genomes. Replication-directed translocations were rare and conservation of synteny was high. The largest difference between C. botulinum group III isolates occurred within their surprisingly large plasmidomes and in the pattern of mobile elements insertions. Five plasmids, constituting 13.5% of the total genetic material, were present in the completed genome. Interestingly, the set of plasmids differed compared to other isolates. The largest plasmid, the botulinum-neurotoxin carrying prophage, was conserved at a level similar to that of the chromosome while the medium-sized plasmids seemed to be undergoing faster genetic drift. These plasmids also contained more mobile elements than other replicons. Several toxins and resistance genes were identified, many of which were located on the plasmids.ConclusionsThe completion of the genome of C. botulinum group III has revealed it to be a genome with dual identity. It belongs to the pathogenic species C. botulinum, but as a genotypic species it should also include C. novyi and C. haemolyticum. The genotypic species share a conserved chromosomal core that can be transformed into various pathogenic variants by modulation of the highly plastic plasmidome.
| S-EPMC3098183 | biostudies-literature
Project description:Animal botulism is mainly associated with Clostridium botulinum group III-producing neurotoxin types C, C/D, D, and D/C. In this report, we present the draft genome sequences of the first five strains of Clostridium botulinum type D/C isolated in Brazil and used for vaccination purposes.
| S-EPMC5442381 | biostudies-literature
Project description:A nontoxigenic strain isolated from a fatal human case of bacterial sepsis was identified as a Clostridium strain from Clostridium botulinum group III, based on the phenotypic characters and 16S rRNA gene sequence, and was found to be related to the mosaic C. botulinum D/C strain according to a multilocus sequence analysis of 5 housekeeping genes.
| S-EPMC3911442 | biostudies-literature
Project description:Here, we report the application of a long-read sequencer, PromethION, for analyzing human cancer genomes. We first conducted whole-genome sequencing on lung cancer cell lines. We found that it is possible to genotype known cancerous mutations, such as point mutations. We also found that long-read sequencing is particularly useful for precisely identifying and characterizing structural aberrations, such as large deletions, gene fusions, and other chromosomal rearrangements. In addition, we identified several medium-sized structural aberrations consisting of complex combinations of local duplications, inversions, and microdeletions. These complex mutations occurred even in key cancer-related genes, such as STK11, NF1, SMARCA4, and PTEN The biological relevance of those mutations was further revealed by epigenome, transcriptome, and protein analyses of the affected signaling pathways. Such structural aberrations were also found in clinical lung adenocarcinoma specimens. Those structural aberrations were unlikely to be reliably detected by conventional short-read sequencing. Therefore, long-read sequencing may contribute to understanding the molecular etiology of patients for whom causative cancerous mutations remain unknown and therapeutic strategies are elusive.
| S-EPMC7545141 | biostudies-literature
Project description:BACKGROUND:Short-read sequencing technologies have made microbial genome sequencing cheap and accessible. However, closing genomes is often costly and assembling short reads from genomes that are repetitive and/or have extreme %GC content remains challenging. Long-read, single-molecule sequencing technologies such as the Oxford Nanopore MinION have the potential to overcome these difficulties, although the best approach for harnessing their potential remains poorly evaluated. RESULTS:We sequenced nine bacterial genomes spanning a wide range of GC contents using Illumina MiSeq and Oxford Nanopore MinION sequencing technologies to determine the advantages of each approach, both individually and combined. Assemblies using only MiSeq reads were highly accurate but lacked contiguity, a deficiency that was partially overcome by adding MinION reads to these assemblies. Even more contiguous genome assemblies were generated by using MinION reads for initial assembly, but these assemblies were more error-prone and required further polishing. This was especially pronounced when Illumina libraries were biased, as was the case for our strains with both high and low GC content. Increased genome contiguity dramatically improved the annotation of insertion sequences and secondary metabolite biosynthetic gene clusters, likely because long-reads can disambiguate these highly repetitive but biologically important genomic regions. CONCLUSIONS:Genome assembly using short-reads is challenged by repetitive sequences and extreme GC contents. Our results indicate that these difficulties can be largely overcome by using single-molecule, long-read sequencing technologies such as the Oxford Nanopore MinION. Using MinION reads for assembly followed by polishing with Illumina reads generated the most contiguous genomes with sufficient accuracy to enable the accurate annotation of important but difficult to sequence genomic features such as insertion sequences and secondary metabolite biosynthetic gene clusters. The combination of Oxford Nanopore and Illumina sequencing can therefore cost-effectively advance studies of microbial evolution and genome-driven drug discovery.
| S-EPMC6325685 | biostudies-literature