scholarly journals Transition From PCR-Ribotyping to Whole Genome Sequencing Based Typing of Clostridioides difficile

2021 ◽  
Vol 11 ◽  
Author(s):  
Helena M. B. Seth-Smith ◽  
Michael Biggel ◽  
Tim Roloff ◽  
Vladimira Hinic ◽  
Thomas Bodmer ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Sequence Data ◽  
Molecular Diagnostic ◽  
Whole Genome ◽  
Clostridioides Difficile ◽  
Challenges And Opportunities ◽  
Long Read ◽  
Illumina Sequence ◽  
Pcr Ribotyping

Clostridioides difficile causes nosocomial outbreaks which can lead to severe and even life-threatening colitis. Rapid molecular diagnostic tests allow the identification of toxin-producing, potentially hypervirulent strains, which is critical for patient management and infection control. PCR-ribotyping has been used for decades as the reference standard to investigate transmission in suspected outbreaks. However, the introduction of whole genome sequencing (WGS) for molecular epidemiology provides a realistic alternative to PCR-ribotyping. In this transition phase it is crucial to understand the strengths and weaknesses of the two technologies, and to assess their correlation. We aimed to investigate ribotype prediction from WGS data, and options for analysis at different levels of analytical granularity. Ribotypes cannot be directly determined from short read Illumina sequence data as the rRNA operons including the ribotype-defining ISR fragments collapse in genome assemblies, and comparison with traditional PCR-ribotyping results becomes impossible. Ribotype extraction from long read Oxford nanopore data also requires optimization. We have compared WGS-based typing with PCR-ribotyping in nearly 300 clinical and environmental isolates from Switzerland, and in addition from the Enterobase database (n=1778). Our results show that while multi-locus sequence type (MLST) often correlates with a specific ribotype, the agreement is not complete, and for some ribotypes the resolution is insufficient. Using core genome MLST (cgMLST) analysis, there is an improved resolution and ribotypes can often be predicted within clusters, using cutoffs of 30-50 allele differences. The exceptions are ribotypes within known ribotype complexes such as RT078/RT106, where the genome differences in cgMLST do not reflect the ribotype segregation. We show that different ribotype clusters display different degrees of diversity, which could be important for the definition of ribotype cluster specific cutoffs. WGS-based analysis offers the ultimate resolution to the SNP level, enabling exploration of patient-to-patient transmission. PCR-ribotyping does not sufficiently discriminate to prove nosocomial transmission with certainty. We discuss the associated challenges and opportunities in a switch to WGS from conventional ribotyping for C. difficile.

scholarly journals Anin silicosurvey ofClostridioides difficileextrachromosomal elements

2019 ◽  
Author(s):  
Bastian Hornung ◽  
Ed J. Kuijper ◽  
Wiep Klaas Smits
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
In Silico ◽  
Sequence Data ◽  
Whole Genome ◽  
Clostridioides Difficile ◽  
Extrachromosomal Elements ◽  
European Nucleotide Archive ◽  
Healthcare Associated ◽  
Systematic Identification

AbstractThe gram-positive enteropathogenClostridioides difficileis the major cause of healthcare associated diarrhoea and is also an important cause of community-acquired infectious diarrhoea. Considering the burden of the disease, many studies have employed whole genome sequencing to identify factors that contribute to virulence and pathogenesis. Though extrachromosomal elements such as plasmids are important for these processes in other bacteria, the few characterized plasmids ofC. difficilehave no relevant functions assigned and no systematic identification of plasmids has been carried out to date. Here, we perform anin silicoanalysis of publicly available sequence data, to show that ∼13% of allC. difficilestrains contain extrachromosomal elements, with 1-6 elements per strain. Our approach identifies known plasmids (e.g. pCD6, pCD630 and cloning plasmids) and 6 novel putative plasmid families. Our study shows that plasmids are abundant and may encode functions that are relevant forC. difficilephysiology. The newly identified plasmids may also form the basis for the construction of novel cloning plasmids forC. difficilethat are compatible with existing tools.RepositoriesThe assembled circular type plasmids have been deposited at the European Nucleotide Archive (ENA) under accession numbers ERZ940801 and ERZ940803-ERZ940808.

scholarly journals Clostridioides difficile Whole-genome Sequencing Differentiates Relapse With the Same Strain From Reinfection With a New Strain

2020 ◽  
Author(s):  
Janice Cho ◽  
Scott Cunningham ◽  
Meng Pu ◽  
Ryan J Lennon ◽  
Jennifer Dens Higano ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Sequence Data ◽  
Infection Prevention And Control ◽  
Whole Genome ◽  
Clostridioides Difficile ◽  
Allelic Differences ◽  
Clostridioides Difficile Infection ◽  
And Control ◽  
Zip Code

Abstract Background Current approaches in tracking Clostridioides difficile infection (CDI) and individualizing patient management are incompletely defined. Methods We recruited 468 subjects with CDI at Mayo Clinic Rochester between May and December 2016 and performed whole-genome sequencing (WGS) on C. difficile isolates from 397. WGS was also performed on isolates from a subset of the subjects at the time of a recurrence of infection. The sequence data were analyzed by determining core genome multilocus sequence type (cgMLST), with isolates grouped by allelic differences and the predicted ribotype. Results There were no correlations between C. difficile isolates based either on cgMLST or ribotype groupings and CDI outcome. An epidemiologic assessment of hospitalized subjects harboring C. difficile isolates with ≤2 allelic differences, based on standard infection prevention and control assessment, revealed no evidence of person-to-person transmission. Interestingly, community-acquired CDI subjects in 40% of groups with ≤2 allelic differences resided within the same zip code. Among 18 subjects clinically classified as having recurrent CDI, WGS revealed 14 with initial and subsequent isolates differing by ≤2 allelic differences, suggesting a relapse of infection with the same initial strain, and 4 with isolates differing by >50 allelic differences, suggesting reinfection. Among the 5 subjects classified as having a reinfection based on the timing of recurrence, 3 had isolates with ≤2 allelic differences between them, suggesting a relapse, and 2 had isolates differing by >50 allelic differences, suggesting reinfection. Conclusions Our findings point to potential transmission of C. difficile in the community. WGS better differentiates relapse from reinfection than do definitions based on the timing of recurrence.

scholarly journals Comprehensive analysis of GBA using a novel algorithm for Illumina whole-genome sequence data or targeted Nanopore sequencing

2021 ◽  
Author(s):  
Marco Toffoli ◽  
Xiao Chen ◽  
Fritz J Sedlazeck ◽  
Chiao-Yin Lee ◽  
Stephen Mullin ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Copy Number ◽  
Sequence Data ◽  
Whole Genome Sequence ◽  
Whole Genome Sequencing Data ◽  
Whole Genome ◽  
Short Read ◽  
Increased Risk ◽  
Long Read

GBA variants cause the autosomal recessive Gaucher disease, and carriers are at increased risk of Parkinson disease (PD) and Lewy body dementia (LBD). The presence of a highly homologous nearby pseudogene (GBAP1) predisposes to a range of structural variants arising from either gene conversion or reciprocal recombination, the latter resulting in copy number gains or losses, complicating genetic testing and analysis. To date, short-read sequencing has not been able to fully resolve these or other variants in the key homology region, and targeted long-read sequencing has not previously resolved reciprocal recombinants. We present and validate two independent methods to resolve recombinant alleles and other variants in GBA: Gauchian, a novel bioinformatics tool for short-read, whole-genome sequencing data analysis, and Oxford Nanopore long-read sequencing after enrichment with appropriate PCR. The methods were concordant for 42 samples including 30 with a range of recombinants and GBAP1-related mutations, and Gauchian outperforms the GATK Best Practices pipeline. Applying Gauchian to Illumina sequencing of over 10,000 individuals from publicly available cohorts shows that copy number variants (CNVs) spanning GBAP1 are relatively common in Africans. CNV frequencies in PD and LBD are similar to controls, but gains may coexist with other mutations in patients, and a modifying effect cannot be excluded. Gauchian detects a higher frequency of GBA variants in LBD than PD, especially severe ones. These findings highlight the importance of accurate GBA mutation detection in these patients, which is possible by either Gauchian analysis of short-read whole genome sequencing, or targeted long-read sequencing.

Detecting a long insertion variant in SAMD12 by SMRT sequencing: implications of long-read whole-genome sequencing for repeat expansion diseases

2018 ◽  
Vol 64 (3) ◽  
pp. 191-197 ◽  
Author(s):  
Takeshi Mizuguchi ◽  
Tomoko Toyota ◽  
Hiroaki Adachi ◽  
Noriko Miyake ◽  
Naomichi Matsumoto ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Repeat Expansion ◽  
Whole Genome ◽  
Smrt Sequencing ◽  
Long Read

scholarly journals Long-read whole genome sequencing and comparative analysis of six strains of the human pathogen Orientia tsutsugamushi

2018 ◽  
Vol 12 (6) ◽  
pp. e0006566 ◽  
Author(s):  
Elizabeth M. Batty ◽  
Suwittra Chaemchuen ◽  
Stuart Blacksell ◽  
Allen L. Richards ◽  
Daniel Paris ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Orientia Tsutsugamushi ◽  
Human Pathogen ◽  
Long Read

scholarly journals Genome Sequencing of Polydrug-, Multidrug-, and Extensively Drug-Resistant Mycobacterium tuberculosis Strains from South India

2019 ◽  
Vol 8 (12) ◽  
Author(s):  
Sivakumar Shanmugam ◽  
Narender Kumar ◽  
Dina Nair ◽  
Mohan Natrajan ◽  
Srikanth Prasad Tripathy ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
South India ◽  
Sequence Data ◽  
Whole Genome ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Content Type ◽  
Extensively Drug Resistant

The genomes of 16 clinical Mycobacterium tuberculosis isolates were subjected to whole-genome sequencing to identify mutations related to resistance to one or more anti-Mycobacterium drugs. The sequence data will help in understanding the genomic characteristics of M. tuberculosis isolates and their resistance mutations prevalent in South India.

scholarly journals Whole Genome Sequencing Resource of the European Larch Canker Pathogen Lachnellula willkommii for Molecular Diagnostic Marker Development

2020 ◽  
Vol 110 (7) ◽  
pp. 1255-1259
Author(s):  
Emily Giroux ◽  
Guillaume J. Bilodeau
Keyword(s):  
United States ◽  
North America ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Draft Genome ◽  
The United States ◽  
Molecular Diagnostic ◽  
Whole Genome ◽  
European Larch ◽  
The North

The filamentous ascomycete fungus Lachnellula willkommii is the causal agent of European larch canker (ELC), one of the most destructive diseases of larch in Europe and a regulated plant pathogen of quarantine significance in Canada and the United States. L. willkommii was first detected in Massachusetts, North America in 1927 on a larch plantation cultivated with nursery stock imported from Great Britain. Despite the decades of practices aimed at eliminating the pathogen, it has reappeared in coastal areas of Canada and the United States. There is concern ELC could spread throughout the range of eastern larch, a transcontinental species typical of the Boreal forest that spans the North American landscape. There is geographic range overlap between several nonpathogenic indigenous Lachnellula species and the reported distribution of L. willkommii in North America. Morphological and biological methods to distinguish L. willkommii are often inadequate as the fungus does not always produce the phenotypic structures that distinguish it from these other saprophytic Lachnellula species. Whole genome sequencing technologies were used to obtain the draft genome sequences of L. willkommii and six other Lachnellula species. Molecular markers identified from the genomic data may be used to discriminate L. willkommii from its nonpathogenic relatives.

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