Closing Clostridium botulinum Group III Genomes Using Long-Read Sequencing.
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ABSTRACT:
SUBMITTER: Woudstra C
PROVIDER: S-EPMC8354527 | biostudies-literature | 2021 Jun
REPOSITORIES: biostudies-literature
Publications
Closing Clostridium botulinum Group III Genomes Using Long-Read Sequencing.
Woudstra Cedric C Mäklin Tommi T Derman Yagmur Y Bano Luca L Skarin Hanna H Mazuet Christelle C Honkela Antti A Lindström Miia M
Microbiology resource announcements 20210603 22
Clostridium botulinum group III is the anaerobic Gram-positive bacterium producing the deadly neurotoxin responsible for animal botulism. Here, we used long-read sequencing to produce four complete genomes from Clostridium botulinum group III neurotoxin types C, D, C/D, and D/C. The protocol for obtaining high-molecular-weight DNA from C. botulinum group III is described. ...[more]
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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.
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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.
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