Whole-Genome Sequencing for Bacterial Strain Typing Using the iSeq100 Platform

Background: Hospital-acquired infections pose a significant threat to patient health. Laboratories are starting to consider whole-genome sequencing (WGS) as a molecular method for outbreak detection and epidemiological surveillance. The objective of this study was to assess the use of the iSeq100 platform (Illumina, San Diego, CA) for accurate sequencing and WGS-based outbreak detection using the bioMérieux EPISEQ CS, a novel cloud-based software for sequence assembly and data analysis. Methods: In total, 25 isolates, including 19 MRSA isolates and 6 ATCC strains were evaluated in this study: A. baumannii ATCC 19606, B. cepacia ATCC 25416, E. faecalis ATCC 29212, E. coli ATCC 25922, P. aeruginosa ATCC 27853 and S. aureus ATCC 25923. DNA extraction of all isolates was performed on the QIAcube (Qiagen, Hilden, Germany) using the DNEasy Ultra Clean Microbial kit extraction protocol. DNA libraries were prepared for WGS using the Nextera DNA Flex Library Prep Kit (Illumina) and sequenced at 2×150-bp on the iSeq100 according to the manufacturer’s instructions. The 19 MRSA isolates were previously characterized by the DiversiLab system (bioMérieux, France). Upon validation of the iSeq100 platform, a new outbreak analysis was performed using WGS analysis using EPISEQ CS. ATCC sequences were compared to assembled reference genomes from the NCBI GenBank to assess the accuracy of the iSeq100 platform. The FASTQ files were aligned via BowTie2 version 2.2.6 software, using default parameters, and FreeBayes version 1.1.0.46-0 was used to call homozygous single-nucleotide polymorphisms (SNPs) with a minimum coverage of 5 and an allele frequency of 0.87 using default parameters. ATCC sequences were analyzed using ResFinder version 3.2 and were compared in silico to the reference genome. Results: EPISEQ CS classified 8 MRSA isolates as unrelated and grouped 11 isolates into 2 separate clusters: cluster A (5 isolates) and cluster B (6 isolates) with similarity scores of ≥99.63% and ≥99.50%, respectively. This finding contrasted with the previous characterization by DiversiLab, which identified 3 clusters of 2, 8, and 11 isolates, respectively. The EPISEQ CS resistome data detected the mecA gene in 18 of 19 MRSA isolates. Comparative analysis of the ATCCsequences to the reference genomes showed 99.9986% concordance of SNPs and 100.00% concordance between the resistance genes present. Conclusions: The iSeq100 platform accurately sequenced the bacterial isolates and could be an affordable alternative in conjunction with EPISEQ CS for epidemiological surveillance analysis and infection prevention.Funding: NoneDisclosures: None

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Loss and Gain in the Evolution of theSalmonella entericaSerovar Gallinarum Biovar Pullorum Genome

mSphere ◽

10.1128/msphere.00627-18 ◽

2019 ◽

Vol 4 (2) ◽

Cited By ~ 6

Author(s):

Yachen Hu ◽

Zhenyu Wang ◽

Bin Qiang ◽

Yaohui Xu ◽

Xiang Chen ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genomic Analysis ◽

Developed Countries ◽

Epidemiological Surveillance ◽

Recent Common Ancestor ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Content Type ◽

Most Recent Common Ancestor

ABSTRACTSalmonella entericasubspeciesentericaserovar Gallinarum biovar Pullorum (S. Pullorum) is the etiological agent of pullorum disease, causing white diarrhea with high mortality in chickens. There are many unsolved issues surrounding the epidemiology ofS. Pullorum, including its origin and transmission history as well as the discordance between its phenotypic heterogeneity and genetic monomorphism. In this paper, we report the results of whole-genome sequencing of a panel of 97S. Pullorum strains isolated between 1962 and 2014 from four countries across three continents. We utilized 6,795 core genome single nucleotide polymorphisms (SNPs) to reconstruct a phylogenetic tree within a spatiotemporal Bayesian framework, estimating that the most recent common ancestor ofS. Pullorum emerged in ∼914 CE (95% confidence interval [95%CI], 565 to 1273 CE). The extantS. Pullorum strains can be divided into four distinct lineages, each of which is significantly associated with geographical distribution. The intercontinental transmissions of lineages III and IV can be traced to the mid-19th century and are probably related to the “Hen Fever” prevalent at that time. Further genomic analysis indicated that the loss or pseudogenization of functional genes involved in metabolism and virulence inS. Pullorum has been ongoing since before and after divergence from the ancestor. In contrast, multiple prophages and plasmids have been acquired byS. Pullorum, and these have endowed it with new characteristics, especially the multidrug resistance conferred by two large plasmids in lineage I. The results of this study provide insight into the evolution ofS. Pullorum and prove the efficiency of whole-genome sequencing in epidemiological surveillance of pullorum disease.IMPORTANCEPullorum disease, an acute poultry septicemia caused bySalmonellaGallinarum biovar Pullorum, is fatal for young chickens and is a heavy burden on poultry industry. The pathogen is rare in most developed countries but still extremely difficult to eliminate in China. Efficient epidemiological surveillance necessitates clarifying the origin of the isolates from different regions and their phylogenic relationships. Genomic epidemiological analysis of 97S. Pullorum strains was carried out to reconstruct the phylogeny and transmission history ofS. Pullorum. Further analysis demonstrated that functional gene loss and acquisition occurred simultaneously throughout the evolution ofS. Pullorum, both of which reflected adaptation to the changing environment. The result of our study will be helpful in surveillance and prevention of pullorum disease.

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Utility of Whole-Genome Sequencing of Escherichia coli O157 for Outbreak Detection and Epidemiological Surveillance

Journal of Clinical Microbiology ◽

10.1128/jcm.01066-15 ◽

2015 ◽

Vol 53 (11) ◽

pp. 3565-3573 ◽

Cited By ~ 48

Author(s):

Anne Holmes ◽

Lesley Allison ◽

Melissa Ward ◽

Timothy J. Dallman ◽

Richard Clark ◽

...

Keyword(s):

Escherichia Coli ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Outbreak Detection ◽

Epidemiological Surveillance ◽

Personal Genome ◽

Whole Genome ◽

Content Type ◽

E Coli ◽

Antimicrobial Resistance Genes

Detailed laboratory characterization ofEscherichia coliO157 is essential to inform epidemiological investigations. This study assessed the utility of whole-genome sequencing (WGS) for outbreak detection and epidemiological surveillance ofE. coliO157, and the data were used to identify discernible associations between genotypes and clinical outcomes. One hundred fiveE. coliO157 strains isolated over a 5-year period from human fecal samples in Lothian, Scotland, were sequenced with the Ion Torrent Personal Genome Machine. A total of 8,721 variable sites in the core genome were identified among the 105 isolates; 47% of the single nucleotide polymorphisms (SNPs) were attributable to six “atypical”E. coliO157 strains and included recombinant regions. Phylogenetic analyses showed that WGS correlated well with the epidemiological data. Epidemiological links existed between cases whose isolates differed by three or fewer SNPs. WGS also correlated well with multilocus variable-number tandem repeat analysis (MLVA) typing data, with only three discordant results observed, all among isolates from cases not known to be epidemiologically related. WGS produced a better-supported, higher-resolution phylogeny than MLVA, confirming that the method is more suitable for epidemiological surveillance ofE. coliO157. A combination ofinsilicoanalyses (VirulenceFinder, ResFinder, and local BLAST searches) were used to determinestxsubtypes, multilocus sequence types (15 loci), and the presence of virulence and acquired antimicrobial resistance genes. There was a high level of correlation between the WGS data and our routine typing methods, although some discordant results were observed, mostly related to the limitation of short sequence read assembly. The data were used to identify sublineages and clades ofE. coliO157, and when they were correlated with the clinical outcome data, they showed that one clade, Ic3, was significantly associated with severe disease. Together, the results show that WGS data can provide higher resolution of the relationships betweenE. coliO157 isolates than that provided by MLVA. The method has the potential to streamline the laboratory workflow and provide detailed information for the clinical management of patients and public health interventions.

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Monitoring meticillin resistant Staphylococcus aureus and its spread in Copenhagen, Denmark, 2013, through routine whole genome sequencing

Eurosurveillance ◽

10.2807/1560-7917.es2015.20.17.21112 ◽

2015 ◽

Vol 20 (17) ◽

Cited By ~ 19

Author(s):

M D Bartels ◽

H Larner-Svensson ◽

H Meiniche ◽

K Kristoffersen ◽

K Schønning ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Protein A ◽

Epidemiological Data ◽

Epidemiological Surveillance ◽

Whole Genome ◽

Staphylococcal Protein A ◽

Hospital Acquired ◽

Type 304

Typing of meticillin resistant Staphylococcus aureus (MRSA) by whole genome sequencing (WGS) is performed routinely in Copenhagen since January 2013. We describe the relatedness, based on WGS data and epidemiological data, of 341 MRSA isolates. These comprised all MRSA (n?=?300) identified in Copenhagen in the first five months of 2013. Moreover, because MRSA of staphylococcal protein A (spa)-type 304 (t304), sequence type (ST) 6 had been associated with a continuous neonatal ward outbreak in Copenhagen starting in 2011, 41 t304 isolates collected in the city between 2010 and 2012 were also included. Isolates from 2013 found to be of t304, ST6 (n=14) were compared to the 41 earlier isolates. In the study, isolates of clonal complex (CC) 22 were examined in detail, as this CC has been shown to include the hospital-acquired epidemic MRSA (EMRSA-15) clone. Finally, all MRSA ST80 were also further analysed, as representatives of an important community-acquired MRSA in Europe. Overall the analysis identified 85 spa-types and 35 STs from 17 CCs. WGS confirmed the relatedness of epidemiologically linked t304 neonatal outbreak isolates. Several non-outbreak related patients had isolates closely related to the neonatal isolates suggesting unrecognised community chains of transmission and insufficient epidemiological data. Only four CC22 isolates were related to EMRSA-15. No community spread was observed among the 13 ST80 isolates. WGS successfully replaced conventional typing and added information to epidemiological surveillance. Creation of a MRSA database allows clustering of isolates based on single nucleotide polymorphism (SNP) calling and has improved our understanding of MRSA transmission.

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Evaluation of Whole Genome Sequencing for outbreak detection of Salmonella enterica serovar Enteritidis

10.26226/morressier.5f4ccc57648d3d8b3f362bad ◽

2020 ◽

Author(s):

Ainhoa Arrieta-Gisasola ◽

Aitor Atxaerandio Landa ◽

Javier Garaizar ◽

Joseba Bikandi ◽

José Karkamo ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Salmonella Enterica ◽

Outbreak Detection ◽

Whole Genome ◽

Salmonella Enterica Serovar Enteritidis

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Whole-genome sequencing reveals rare off-target mutations in CRISPR/Cas9-edited grapevine

Horticulture Research ◽

10.1038/s41438-021-00549-4 ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Xianhang Wang ◽

Mingxing Tu ◽

Ya Wang ◽

Wuchen Yin ◽

Yu Zhang ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Editing ◽

Genome Sequencing ◽

Plant Biotechnology ◽

High Specificity ◽

Fruit Trees ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Indel Mutation ◽

Target Sites

AbstractThe CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) system is a powerful tool for targeted genome editing, with applications that include plant biotechnology and functional genomics research. However, the specificity of Cas9 targeting is poorly investigated in many plant species, including fruit trees. To assess the off-target mutation rate in grapevine (Vitis vinifera), we performed whole-genome sequencing (WGS) of seven Cas9-edited grapevine plants in which one of two genes was targeted by CRISPR/Cas9 and three wild-type (WT) plants. In total, we identified between 202,008 and 272,397 single nucleotide polymorphisms (SNPs) and between 26,391 and 55,414 insertions/deletions (indels) in the seven Cas9-edited grapevine plants compared with the three WT plants. Subsequently, 3272 potential off-target sites were selected for further analysis. Only one off-target indel mutation was identified from the WGS data and validated by Sanger sequencing. In addition, we found 243 newly generated off-target sites caused by genetic variants between the Thompson Seedless cultivar and the grape reference genome (PN40024) but no true off-target mutations. In conclusion, we observed high specificity of CRISPR/Cas9 for genome editing of grapevine.

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Risk prediction and marker selection in nonsynonymous single nucleotide polymorphisms using whole genome sequencing data

Animal Cells and Systems ◽

10.1080/19768354.2020.1860125 ◽

2020 ◽

Vol 24 (6) ◽

pp. 321-328

Author(s):

Young-Sup Lee ◽

KyeongHye Won ◽

Donghyun Shin ◽

Jae-Don Oh

Keyword(s):

Single Nucleotide Polymorphisms ◽

Whole Genome Sequencing ◽

Risk Prediction ◽

Genome Sequencing ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Sequencing Data ◽

Single Nucleotide ◽

Marker Selection

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A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing

Scientific Reports ◽

10.1038/s41598-020-80031-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kathy E. Raven ◽

Sophia T. Girgis ◽

Asha Akram ◽

Beth Blane ◽

Danielle Leek ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Dna Extraction ◽

Genome Sequencing ◽

Clinical Microbiology ◽

Bacterial Species ◽

Outbreak Detection ◽

Whole Genome ◽

Library Preparation ◽

Simultaneous Processing ◽

Antimicrobial Resistance Gene

AbstractWhole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.

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Fast genetic mapping using insertion-deletion polymorphisms in Caenorhabditis elegans

Scientific Reports ◽

10.1038/s41598-021-90190-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ho-Yon Hwang ◽

Jiou Wang

Keyword(s):

Caenorhabditis Elegans ◽

Genetic Mapping ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genetic Material ◽

Mapping Method ◽

Forward Genetics ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Large Populations

AbstractGenetic mapping is used in forward genetics to narrow the list of candidate mutations and genes corresponding to the mutant phenotype of interest. Even with modern advances in biology such as efficient identification of candidate mutations by whole-genome sequencing, mapping remains critical in pinpointing the responsible mutation. Here we describe a simple, fast, and affordable mapping toolkit that is particularly suitable for mapping in Caenorhabditis elegans. This mapping method uses insertion-deletion polymorphisms or indels that could be easily detected instead of single nucleotide polymorphisms in commonly used Hawaiian CB4856 mapping strain. The materials and methods were optimized so that mapping could be performed using tiny amount of genetic material without growing many large populations of mutants for DNA purification. We performed mapping of previously known and unknown mutations to show strengths and weaknesses of this method and to present examples of completed mapping. For situations where Hawaiian CB4856 is unsuitable, we provide an annotated list of indels as a basis for fast and easy mapping using other wild isolates. Finally, we provide rationale for using this mapping method over other alternatives as a part of a comprehensive strategy also involving whole-genome sequencing and other methods.

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Mycobacterium chimaera genomics with regard to epidemiological and clinical investigations conducted for the open-chest post-surgical Mycobacterium chimaera infections outbreak

Open Forum Infectious Diseases ◽

10.1093/ofid/ofab192 ◽

2021 ◽

Author(s):

Emmanuel Lecorche ◽

Côme Daniau ◽

Kevin La ◽

Faiza Mougari ◽

Hanaa Benmansour ◽

...

Keyword(s):

Single Nucleotide Polymorphisms ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Clinical Isolates ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Healthcare Facilities ◽

Open Chest

Abstract Background Post-surgical infections due to Mycobacterium chimaera appeared as a novel nosocomial threat in 2015, with a worldwide outbreak due to contaminated heater-cooler units used in open chest surgery. We report the results of investigations conducted in France including whole genome sequencing comparison of patient and HCU isolates. Methods We sought M. chimaera infection cases from 2010 onwards through national epidemiological investigations in healthcare facilities performing cardiopulmonary bypass together with a survey on good practices and systematic heater-cooler unit microbial analyses. Clinical and HCU isolates were subjected to whole genome sequencing analyzed with regards to the reference outbreak strain Zuerich-1. Results Only two clinical cases were shown to be related to the outbreak, although 23% (41/175) heater-cooler units were declared positive for M. avium complex. Specific measures to prevent infection were applied in 89% (50/56) healthcare facilities although only 14% (8/56) of them followed the manufacturer maintenance recommendations. Whole genome sequencing comparison showed that the clinical isolates and 72% (26/36) of heater-cooler unit isolates belonged to the epidemic cluster. Within clinical isolates, 5 to 9 non-synonymous single nucleotide polymorphisms were observed, among which an in vivo mutation in a putative efflux pump gene observed in a clinical isolate obtained for one patient under antimicrobial treatment. Conclusions Cases of post-surgical M. chimaera infections were declared to be rare in France, although heater-cooler units were contaminated as in other countries. Genomic analyses confirmed the connection to the outbreak and identified specific single nucleotide polymorphisms, including one suggesting fitness evolution in vivo.

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