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

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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Illumina DNA Prep (M) Tagmentation Library Preparation for use on an Illumina MiSeq Sequencer v2

10.17504/protocols.io.bx4qpqvw ◽

2021 ◽

Author(s):

Julie Haendiges ◽

Narjol Gonzalez-Escalona ◽

Ruth E Timme ◽

Maria Balkey

Keyword(s):

Whole Genome Sequencing ◽

Dna Extraction ◽

Genome Sequencing ◽

Illumina Miseq ◽

Whole Genome ◽

Library Preparation ◽

High Quality ◽

Dna Quantitation ◽

Quality Checks ◽

Automated Dna Extraction

This procedure outlines the protocol for whole genome sequencing of bacterial organisms using the Illumina DNA Prep library preparation kit for sequencing on an Illumina MiSeq sequencer. This document applies to all laboratory personnel in the Division of Microbiology (DM) as well as laboratories in the GenomeTrakr Network. Complete in order: 1. DNA Extraction (Manual DNA Extraction or Automated DNA Extraction using the Qiacube) Step-by-step procedures to obtain high quality DNA from isolates in TSB for whole genome sequencing 2. DNA Quantitation Quantitation of extracted DNA using the Qubit Flourometer 3. Library Preparation for WGS (Included SOP or Library Preparation using Illumina Nextera XT ) Library preparation using NexteraXT or Illumina DNA Prep (previously Nextera DNA Flex) 4. Sequencing using Illumina MiSeq 5. Data Quality Checks and NCBI Submission

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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 for Bacterial Strain Typing Using the iSeq100 Platform

Infection Control and Hospital Epidemiology ◽

10.1017/ice.2020.1098 ◽

2020 ◽

Vol 41 (S1) ◽

pp. s434-s434

Author(s):

Grant Vestal ◽

Steven Bruzek ◽

Amanda Lasher ◽

Amorce Lima ◽

Suzane Silbert

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Outbreak Detection ◽

Epidemiological Surveillance ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Dna Libraries ◽

Patient Health ◽

Hospital Acquired ◽

Reference Genomes

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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Evaluation of Whole Genome Sequencing for Outbreak Detection of Salmonella enterica

PLoS ONE ◽

10.1371/journal.pone.0087991 ◽

2014 ◽

Vol 9 (2) ◽

pp. e87991 ◽

Cited By ~ 154

Author(s):

Pimlapas Leekitcharoenphon ◽

Eva M. Nielsen ◽

Rolf S. Kaas ◽

Ole Lund ◽

Frank M. Aarestrup

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Salmonella Enterica ◽

Outbreak Detection ◽

Whole Genome

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Transmission of ESBL-producing Enterobacteriaceae and their mobile genetic elements—identification of sources by whole genome sequencing: study protocol for an observational study in Switzerland

BMJ Open ◽

10.1136/bmjopen-2018-021823 ◽

2018 ◽

Vol 8 (2) ◽

pp. e021823 ◽

Cited By ~ 12

Author(s):

Tanja Stadler ◽

Dominik Meinel ◽

Lisandra Aguilar-Bultet ◽

Jana S Huisman ◽

Ruth Schindler ◽

...

Keyword(s):

Observational Study ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Bacterial Species ◽

Mobile Genetic Elements ◽

University Hospital ◽

Whole Genome ◽

Hospital Acquired Infections ◽

Genetic Elements ◽

Hospital Acquired

IntroductionExtended-spectrum beta-lactamases (ESBL)-producing Enterobacteriaceae were first described in relation with hospital-acquired infections. In the 2000s, the epidemiology of ESBL-producing organisms changed as especially ESBL-producingEscherichia coliwas increasingly described as an important cause of community-acquired infections, supporting the hypothesis that in more recent years ESBL-producing Enterobacteriaceae have probably been imported into hospitals rather than vice versa. Transmission of ESBL-producing Enterobacteriaceae is complicated by ESBL genes being encoded on self-transmissible plasmids, which can be exchanged among the same and different bacterial species. The aim of this research project is to quantify hospital-wide transmission of ESBL-producing Enterobacteriaceae on both the level of bacterial species and the mobile genetic elements and to determine if hospital-acquired infections caused by ESBL producers are related to strains and mobile genetic elements predominantly circulating in the community or in the healthcare setting. This distinction is critical in prevention since the former emphasises the urgent need to establish or reinforce antibiotic stewardship programmes, and the latter would call for more rigorous infection control.Methods and analysisThis protocol presents an observational study that will be performed at the University Hospital Basel and in the city of Basel, Switzerland. ESBL-producing Enterobacteriaceae will be collected from any specimens obtained by routine clinical practice or by active screening in both inpatient and outpatient settings, as well as from wastewater samples and foodstuffs, both collected monthly over a 12-month period for analyses by whole genome sequencing. Bacterial chromosomal, plasmid and ESBL-gene sequences will be compared within the cohort to determine genetic relatedness and migration between humans and their environment.Ethics and disseminationThis study has been approved by the local ethics committee (Ethikkommission Nordwest-und Zentralschweiz) as a quality control project (Project-ID 2017–00100). The results of this study will be published in peer-reviewed medical journals, communicated to participants, the general public and all relevant stakeholders.

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Rapid, high throughput library preparation of SARS-CoV2 using Illumina’s DNA Prep Library Preparation Kit v2

10.17504/protocols.io.b3vgqn3w ◽

2022 ◽

Author(s):

Jason Nguyen ◽

Rebecca Hickman ◽

Tracy Lee ◽

Natalie Prystajecky ◽

John Tyson

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

High Throughput ◽

Illumina Miseq ◽

Whole Genome ◽

Library Preparation ◽

Dna Libraries ◽

Reaction Volumes ◽

Single Size ◽

Stop Buffer

This procedure provides instructions on how to prepare DNA libraries for whole genome sequencing on an Illumina MiSeq or NextSeq using Illumina’s DNA Prep Library Preparation Kit scaled to half reaction volumes with modifications to the post-PCR procedures; tagmentation stop buffer and associated washes are removed and libraries are pooled post PCR then a single size selection is performed. This protocol is used to sequence SARS-CoV-2 using the cDNA/PCR protocol: https://dx.doi.org/10.17504/protocols.io.b3viqn4e

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Whole-Genome Sequencing: Automated, Nonindexed Library Preparation

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot094623 ◽

2016 ◽

Vol 2017 (3) ◽

pp. pdb.prot094623 ◽

Cited By ~ 4

Author(s):

Elaine Mardis ◽

W. Richard McCombie

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome ◽

Library Preparation

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Spill-Over from Public Health? First Detection of an OXA-48-Producing Escherichia coli in a German Pig Farm

Microorganisms ◽

10.3390/microorganisms8060855 ◽

2020 ◽

Vol 8 (6) ◽

pp. 855 ◽

Cited By ~ 2

Author(s):

Alexandra Irrgang ◽

Natalie Pauly ◽

Bernd-Alois Tenhagen ◽

Mirjam Grobbel ◽

Annemarie Kaesbohrer ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Bacterial Species ◽

Multidrug Resistant ◽

Meat Production ◽

Whole Genome ◽

Phenotypic Resistance ◽

Long Read ◽

Pig Farm ◽

Human Infections

Resistance to carbapenems is a severe threat to human health. These last resort antimicrobials are indispensable for the treatment of severe human infections with multidrug-resistant Gram-negative bacteria. In accordance with their increasing medical impact, carbapenemase-producing Enterobacteriaceae (CPE) might be disseminated from colonized humans to non-human reservoirs (i.e., environment, animals, food). In Germany, the occurrence of CPE in livestock and food has been systematically monitored since 2016. In the 2019 monitoring, an OXA-48-producing E. coli (19-AB01443) was recovered from a fecal sample of a fattening pig. Phenotypic resistance was confirmed by broth microdilution and further characterized by PFGE, conjugation, and combined short-/long-read whole genome sequencing. This is the first detection of this resistance determinant in samples from German meat production. Molecular characterization and whole-genome sequencing revealed that the blaOXA-48 gene was located on a common pOXA-48 plasmid-prototype. This plasmid-type seems to be globally distributed among various bacterial species, but it was frequently associated with clinical Klebsiella spp. isolates. Currently, the route of introduction of this plasmid/isolate combination into the German pig production is unknown. We speculate that due to its strong correlation with human isolates a transmission from humans to livestock has occurred.

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Mycobacterial DNA Extraction for Whole-Genome Sequencing from Early Positive Liquid (MGIT) Cultures

Journal of Clinical Microbiology ◽

10.1128/jcm.03073-14 ◽

2015 ◽

Vol 53 (4) ◽

pp. 1137-1143 ◽

Cited By ~ 47

Author(s):

Antonina A. Votintseva ◽

Louise J. Pankhurst ◽

Luke W. Anson ◽

Marcus R. Morgan ◽

Deborah Gascoyne-Binzi ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Dna Extraction ◽

Genome Sequencing ◽

Solid Phase ◽

Clinical Samples ◽

Reference Laboratory ◽

Whole Genome ◽

Mycobacterial Species ◽

Accurate Identification ◽

Content Type

We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ≥0.2 ng/μl of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with <5% of reads derived from human or nasopharyngeal flora for 88% and 91% of samples, respectively. A total of 59 (98%) of 60 samples that were identified by the national mycobacterial reference laboratory (NMRL) asMycobacterium tuberculosiswere successfully mapped to the H37Rv reference, with >90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools.

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Discordant bioinformatic predictions of antimicrobial resistance from whole-genome sequencing data of bacterial isolates: An inter-laboratory study

10.1101/793885 ◽

2019 ◽

Cited By ~ 1

Author(s):

Ronan M. Doyle ◽

Denise M. O’Sullivan ◽

Sean D. Aller ◽

Sebastian Bruchmann ◽

Taane Clark ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Laboratory Study ◽

Clinical Microbiology ◽

Sequence Data ◽

Clinical Samples ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Sequencing Data

AbstractBackgroundAntimicrobial resistance (AMR) poses a threat to public health. Clinical microbiology laboratories typically rely on culturing bacteria for antimicrobial susceptibility testing (AST). As the implementation costs and technical barriers fall, whole-genome sequencing (WGS) has emerged as a ‘one-stop’ test for epidemiological and predictive AST results. Few published comparisons exist for the myriad analytical pipelines used for predicting AMR. To address this, we performed an inter-laboratory study providing sets of participating researchers with identical short-read WGS data sequenced from clinical isolates, allowing us to assess the reproducibility of the bioinformatic prediction of AMR between participants and identify problem cases and factors that lead to discordant results.MethodsWe produced ten WGS datasets of varying quality from cultured carbapenem-resistant organisms obtained from clinical samples sequenced on either an Illumina NextSeq or HiSeq instrument. Nine participating teams (‘participants’) were provided these sequence data without any other contextual information. Each participant used their own pipeline to determine the species, the presence of resistance-associated genes, and to predict susceptibility or resistance to amikacin, gentamicin, ciprofloxacin and cefotaxime.ResultsIndividual participants predicted different numbers of AMR-associated genes and different gene variants from the same clinical samples. The quality of the sequence data, choice of bioinformatic pipeline and interpretation of the results all contributed to discordance between participants. Although much of the inaccurate gene variant annotation did not affect genotypic resistance predictions, we observed low specificity when compared to phenotypic AST results but this improved in samples with higher read depths. Had the results been used to predict AST and guide treatment a different antibiotic would have been recommended for each isolate by at least one participant.ConclusionsWe found that participants produced discordant predictions from identical WGS data. These challenges, at the final analytical stage of using WGS to predict AMR, suggest the need for refinements when using this technology in clinical settings. Comprehensive public resistance sequence databases and standardisation in the comparisons between genotype and resistance phenotypes will be fundamental before AST prediction using WGS can be successfully implemented in standard clinical microbiology laboratories.

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