Whole-Genome Sequencing for Rapid and Accurate Identification of Bacterial Transmission Pathways

Members of the genus Staphylococcus have been isolated from humans, animals, and the environment. Accurate identification with whole-genome sequencing requires access to data derived from type strains.

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Integration of Whole-Genome Sequencing into Infection Control Practices: the Potential and the Hurdles

Journal of Clinical Microbiology ◽

10.1128/jcm.00349-15 ◽

2015 ◽

Vol 53 (4) ◽

pp. 1054-1055 ◽

Cited By ~ 10

Author(s):

Elizabeth Robilotti ◽

Mini Kamboj

Keyword(s):

Infection Control ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Clinical Epidemiology ◽

Great Promise ◽

Whole Genome ◽

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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A Bioinformatics Pipeline for Estimating Mitochondria DNA Copy Number and Heteroplasmy Levels from Whole Genome Sequencing Data

10.1101/2021.12.28.21268452 ◽

2021 ◽

Author(s):

Stephanie L Battle ◽

Daniela Puiu ◽

Eric Boerwinkle ◽

Kent Taylor ◽

Jerome Rotter ◽

...

Keyword(s):

Mitochondrial Genome ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Copy Number ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Dna Molecules ◽

Sequencing Data ◽

Bioinformatics Pipeline ◽

Accurate Identification

Mitochondrial diseases are a heterogeneous group of disorders that can be caused by mutations in the nuclear or mitochondrial genome. Mitochondrial DNA variants may exist in a state of heteroplasmy, where a percentage of DNA molecules harbor a variant, or homoplasmy, where all DNA molecules have a variant. The relative quantity of mtDNA in a cell, or copy number (mtDNA-CN), is associated with mitochondrial function, human disease, and mortality. To facilitate accurate identification of heteroplasmy and quantify mtDNA-CN, we built a bioinformatics pipeline that takes whole genome sequencing data and outputs mitochondrial variants, and mtDNA-CN. We incorporate variant annotations to facilitate determination of variant significance. Our pipeline yields uniform coverage by remapping to a circularized chrM and recovering reads falsely mapped to nuclear-encoded mitochondrial sequences. Notably, we construct a consensus chrM sequence for each sample and recall heteroplasmy against the sample's unique mitochondrial genome. We observe an approximately 3-fold increased association with age for heteroplasmic variants in non-homopolymer regions and, are better able to capture genetic variation in the D-loop of chrM compared to existing software. Our bioinformatics pipeline more accurately captures features of mitochondrial genetics than existing pipelines that are important in understanding how mitochondrial dysfunction contributes to disease.

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Direct Whole-Genome Sequencing of Sputum Accurately Identifies Drug-Resistant Mycobacterium tuberculosis Faster than MGIT Culture Sequencing

Journal of Clinical Microbiology ◽

10.1128/jcm.00666-18 ◽

2018 ◽

Vol 56 (8) ◽

Cited By ~ 47

Author(s):

Ronan M. Doyle ◽

Carrie Burgess ◽

Rachel Williams ◽

Rebecca Gorton ◽

Helen Booth ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Antimicrobial Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Resistance Testing ◽

Whole Genome ◽

Accurate Identification ◽

Content Type ◽

Resistance Profile ◽

Mgit Culture

ABSTRACT The current methods available to diagnose antimicrobial-resistant Mycobacterium tuberculosis infections require a positive culture or only test a limited number of resistance-associated mutations. A rapid accurate identification of antimicrobial resistance enables the prompt initiation of effective treatment. Here, we determine the utility of whole-genome sequencing (WGS) of M. tuberculosis directly from routinely obtained diagnostic sputum samples to provide a comprehensive resistance profile compared to that from mycobacterial growth indicator tube (MGIT) WGS. We sequenced M. tuberculosis from 43 sputum samples by targeted DNA enrichment using the Agilent SureSelectXT kit, and 43 MGIT positive samples from each participant. Thirty two (74%) sputum samples and 43 (100%) MGIT samples generated whole genomes. The times to antimicrobial resistance profiles and concordance were compared with Xpert MTB/RIF and phenotypic resistance testing from cultures of the same samples. Antibiotic susceptibility could be predicted from WGS of sputum within 5 days of sample receipt and up to 24 days earlier than WGS from MGIT culture and up to 31 days earlier than phenotypic testing. Direct sputum results could be reduced to 3 days with faster hybridization and if only regions encoding drug resistance are sequenced. We show that direct sputum sequencing has the potential to provide comprehensive resistance detection significantly faster than MGIT whole-genome sequencing or phenotypic testing of resistance from cultures in a clinical setting. This improved turnaround time enables prompt appropriate treatment with associated patient and health service benefits. Improvements in sample preparation are necessary to ensure comparable sensitivities and complete resistance profile predictions in all cases.

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Adaptation of Oxford Nanopore technology for hepatitis C whole genome sequencing and identification of within-host viral variants

BMC Genomics ◽

10.1186/s12864-021-07460-1 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Nasir Riaz ◽

Preston Leung ◽

Kirston Barton ◽

Martin A. Smith ◽

Shaun Carswell ◽

...

Keyword(s):

Cost Effectiveness ◽

Hepatitis C ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Reference Sequence ◽

Whole Genome ◽

Nanopore Sequencing ◽

Accurate Identification ◽

Low Frequencies ◽

Oxford Nanopore

Abstract Background Hepatitis C (HCV) and many other RNA viruses exist as rapidly mutating quasi-species populations in a single infected host. High throughput characterization of full genome, within-host variants is still not possible despite advances in next generation sequencing. This limitation constrains viral genomic studies that depend on accurate identification of hemi-genome or whole genome, within-host variants, especially those occurring at low frequencies. With the advent of third generation long read sequencing technologies, including Oxford Nanopore Technology (ONT) and PacBio platforms, this problem is potentially surmountable. ONT is particularly attractive in this regard due to the portable nature of the MinION sequencer, which makes real-time sequencing in remote and resource-limited locations possible. However, this technology (termed here ‘nanopore sequencing’) has a comparatively high technical error rate. The present study aimed to assess the utility, accuracy and cost-effectiveness of nanopore sequencing for HCV genomes. We also introduce a new bioinformatics tool (Nano-Q) to differentiate within-host variants from nanopore sequencing. Results The Nanopore platform, when the coverage exceeded 300 reads, generated comparable consensus sequences to Illumina sequencing. Using HCV Envelope plasmids (~ 1800 nt) mixed in known proportions, the capacity of nanopore sequencing to reliably identify variants with an abundance as low as 0.1% was demonstrated, provided the autologous reference sequence was available to identify the matching reads. Successful pooling and nanopore sequencing of 52 samples from patients with HCV infection demonstrated its cost effectiveness (AUD$ 43 per sample with nanopore sequencing versus $100 with paired-end short read technology). The Nano-Q tool successfully separated between-host sequences, including those from the same subtype, by bulk sorting and phylogenetic clustering without an autologous reference sequence (using only a subtype-specific generic reference). The pipeline also identified within-host viral variants and their abundance when the parameters were appropriately adjusted. Conclusion Cost effective HCV whole genome sequencing and within-host variant identification without haplotype reconstruction are potential advantages of nanopore sequencing.

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Integrated patient network and genomic plasmid analysis reveal a regional, multi-species outbreak of carbapenemase-producing Enterobacterales carrying both blaIMP and mcr-9 genes

10.1101/2021.10.28.21265436 ◽

2021 ◽

Author(s):

Adhiratha Boonyasiri ◽

Ashlegh Myall ◽

Yu Wan ◽

Frances Bolt ◽

Alice Ledda ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Large Data ◽

Whole Genome ◽

Plasmid Analysis ◽

Patient Pathways ◽

Hospital Networks ◽

Bacterial Transmission ◽

Real Time Identification ◽

Medical Specialities

The incidence of carbapenemase-producing Enterobacterales (CPE) is rising globally, yet Imipenemase (IMP) carbapenemases remain relatively rare. This study describes an investigation of the emergence of IMP-encoding CPE amongst diverse Enterobacterales species between 2016 and 2019 in patients across a London regional hospital network. A network analysis approach to patient pathways, using routinely collected electronic health records, identified previously unrecognised contacts between patients who were IMP CPE positive on screening, implying potential bacterial transmission events. Whole genome sequencing of 85 Enterobacterales isolates from these patients revealed that 86% (73/85) were diverse species (predominantly Klebsiella spp, Enterobacter spp, E. coli) and harboured an IncHI2 plasmid, which carried both blaIMP and the putative mobile colistin resistance gene mcr-9. Detailed phylogenetic analysis identified two distinct IncHI2 plasmid lineages, A and B, both of which showed significant association with patient movements between four hospital sites and across medical specialities. Combined, our patient network and plasmid analyses demonstrate an interspecies, plasmid-mediated outbreak of blaIMPCPE, which remained unidentified during standard microbiology and infection control investigations. With whole genome sequencing (WGS) technologies and large-data incorporation, the outbreak investigation approach proposed here provides a framework for real-time identification of key factors causing pathogen spread. Analysing outbreaks at the plasmid level reveals that resistance may be wider spread than suspected, allowing more targetted interventions to stop the transmission of resistance within hospital networks.

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