Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses

Staphylococcus aureus (S. aureus) is a common cause of surgical site infections (SSIs) globally. Data on the occurrence of methicillin-susceptible S. aureus (MSSA) as well as methicillin-resistant S. aureus (MRSA) among patients with surgical site infections (SSIs) in sub-Saharan African are scarce. We characterized S. aureus from SSIs in Ghana using molecular methods and antimicrobial susceptibility testing (AST). Wound swabs or aspirate samples were collected from subjects with SSIs. S. aureus was identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF-MS); AST was performed by Kirby-Bauer disk diffusion, and results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guideline. Detection of spa, mecA, and pvl genes was performed by polymerase chain reaction (PCR). Whole-genome sequencing (WGS) was done using the Illumina MiSeq platform. Samples were collected from 112 subjects, with 13 S. aureus isolates recovered. Of these, 92% were sensitive to co-trimoxazole, 77% to clindamycin, and 54% to erythromycin. Multi-drug resistance was detected in 5 (38%) isolates. The four mecA gene-positive MRSA isolates detected belonged to ST152 (n = 3) and ST5 (n = 1). In total, 62% of the isolates were positive for the Panton-Valentine leukocidin (pvl) toxin gene. This study reports, for the first time, a pvl-positive ST152-t355 MRSA clone from SSIs in Ghana. The occurrence of multi-drug-resistant S. aureus epidemic clones suggests that continuous surveillance is required to monitor the spread and resistance trends of S. aureus in hospital settings in the country.

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Generating 1200bp tiled Amplicons for SARS-CoV2 Whole Genome Sequencing v2

10.17504/protocols.io.b3viqn4e ◽

2022 ◽

Author(s):

jason.nguyen not provided ◽

Tracy Lee ◽

Rebecca Hickman ◽

Natalie Prystajecky ◽

John Tyson

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Illumina Miseq ◽

Whole Genome ◽

Oxford Nanopore ◽

Sequencing Platforms

This procedure provides instructions for how to generate amplicons across the entire SARS-CoV-2 genome to be used for downstream whole genome sequencing applications, including Illumina MiSeq/NextSeq or Oxford Nanopore MinION sequencing platforms. The steps involved in this protocol were derived from version 3 of Freed et al protocol nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon)V.3 available at https://dx.doi.org/10.17504/protocols.io.bgggjttw

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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 of Salmonella enterica Serovar Infantis and Kentucky Isolates Obtained from Layer Poultry Farms in Ecuador

Microbiology Resource Announcements ◽

10.1128/mra.00091-20 ◽

2020 ◽

Vol 9 (13) ◽

Author(s):

William Calero-Cáceres ◽

Joyce Villacís ◽

Maria Ishida ◽

Elton Burnett ◽

Christian Vinueza-Burgos

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Salmonella Enterica ◽

Egg Production ◽

Illumina Miseq ◽

Whole Genome ◽

Genome Sequences ◽

Content Type ◽

Poultry Farms

Five strains of Salmonella enterica subsp. enterica serovar Infantis and two strains of S. enterica subsp. enterica serovar Kentucky isolated in 2017 from Ecuadorian layer poultry farms were sequenced using Illumina MiSeq technology. These isolates were collected on layer farms in central Ecuador, one of the most important areas of egg production in the country. The genome sequences of these isolates show valuable information for surveillance purposes.

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Assessment of Metagenomic MinION and Illumina sequencing as an approach for the recovery of whole genome sequences of chikungunya and dengue viruses directly from clinical samples

10.1101/355560 ◽

2018 ◽

Cited By ~ 2

Author(s):

Liana E. Kafetzopoulou ◽

Kyriakos Efthymiadis ◽

Kuiama Lewandowski ◽

Ant Crook ◽

Dan Carter ◽

...

Keyword(s):

Dengue Virus ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Illumina Miseq ◽

Clinical Samples ◽

Whole Genome ◽

Metagenomic Sequencing ◽

Front Line ◽

Genome Sequences ◽

Oxford Nanopore

AbstractThe recent global emergence and re-emergence of arboviruses has caused significant human disease. Common vectors, symptoms and geographical distribution make differential diagnosis both important and challenging. We performed metagenomic sequencing using both the Illumina MiSeq and the portable Oxford Nanopore MinION to study the feasibility of whole genome sequencing from clinical samples containing chikungunya or dengue virus, two of the most important arboviruses. Direct metagenomic sequencing of nucleic acid extracts from serum and plasma without viral enrichment allowed for virus and coinfection identification, subtype determination and in the majority of cases elucidated complete or near-complete genomes adequate for phylogenetic analysis. This work demonstrates that metagenomic whole genome sequencing is feasible for over 90% and 80% of chikungunya and dengue virus PCR-positive patient samples respectively. It confirms the feasibility of field metagenomic sequencing for these and likely other RNA viruses, highlighting the applicability of this approach to front-line public health.

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Same-day diagnostic and surveillance data for tuberculosis via whole genome sequencing of direct respiratory samples

10.1101/094789 ◽

2016 ◽

Cited By ~ 5

Author(s):

Antonina A. Votintseva ◽

Phelim Bradley ◽

Louise Pankhurst ◽

Carlos del Ojo Elias ◽

Matthew Loose ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Antibiotic Susceptibility ◽

Point Of Care ◽

Low Cost ◽

Illumina Miseq ◽

Turnaround Time ◽

Reference Laboratory ◽

Whole Genome ◽

Specific Pcr

AbstractRoutine full characterization of Mycobacterium tuberculosis (TB) is culture-based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near point of care.We demonstrate a low-cost DNA extraction method for TB WGS direct from patient samples. We initially evaluated the method using the Illumina MiSeq sequencer (40 smear-positive respiratory samples, obtained after routine clinical testing, and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction was obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. Using an Illumina MiSeq/MiniSeq the workflow from patient sample to results can be completed in 44/16 hours at a cost of £96/£198 per sample.We then employed a non-specific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis BCG strain (BCG), and to combined culture-negative sputum DNA and BCG DNA. For the latest flowcell, the estimated turnaround time from patient to identification of BCG was 6 hours, with full susceptibility and surveillance results 2 hours later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of the MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis in direct samples.

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Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples

Journal of Clinical Microbiology ◽

10.1128/jcm.02483-16 ◽

2017 ◽

Vol 55 (5) ◽

pp. 1285-1298 ◽

Cited By ~ 169

Author(s):

Antonina A. Votintseva ◽

Phelim Bradley ◽

Louise Pankhurst ◽

Carlos del Ojo Elias ◽

Matthew Loose ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Antibiotic Susceptibility ◽

Point Of Care ◽

Low Cost ◽

Illumina Miseq ◽

Reference Laboratory ◽

Whole Genome ◽

Content Type ◽

Phylogenetic Placement

ABSTRACT Routine full characterization of Mycobacterium tuberculosis is culture based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near the point of care. We demonstrate a low-cost method of DNA extraction directly from patient samples for M. tuberculosis WGS. We initially evaluated the method by using the Illumina MiSeq sequencer (40 smear-positive respiratory samples obtained after routine clinical testing and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction were obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. With Illumina MiSeq/MiniSeq, the workflow from patient sample to results can be completed in 44/16 h at a reagent cost of £96/£198 per sample. We then employed a nonspecific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis strain BCG DNA and to combined culture-negative sputum DNA and BCG DNA. For flow cell version R9.4, the estimated turnaround time from patient to identification of BCG, detection of pyrazinamide resistance, and phylogenetic placement was 7.5 h, with full susceptibility results 5 h later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis DNA in direct samples.

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Identification and Whole-Genome Sequencing of a Monkeypox Virus Strain Isolated in Israel

Microbiology Resource Announcements ◽

10.1128/mra.01524-19 ◽

2020 ◽

Vol 9 (10) ◽

Cited By ~ 2

Author(s):

Inbar Cohen-Gihon ◽

Ofir Israeli ◽

Ohad Shifman ◽

Noam Erez ◽

Sharon Melamed ◽

...

Keyword(s):

West Africa ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Virus Strain ◽

Illumina Miseq ◽

Whole Genome Sequence ◽

Whole Genome ◽

Monkeypox Virus ◽

Oxford Nanopore ◽

Oxford Nanopore Technologies

We report the whole-genome sequence of a monkeypox virus strain isolated in Israel. The strain was isolated in 2018 from a patient travelling back from West Africa. The virus was fully sequenced on the Illumina MiSeq and Oxford Nanopore Technologies MinION platforms.

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864. Whole Genome Sequencing is Unable to Track Candida auris Transmission

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1053 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S470-S471

Author(s):

Scott C Roberts ◽

Egon A Ozer ◽

Teresa Zembower ◽

Chao Qi

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Illumina Miseq ◽

Whole Genome ◽

Single Nucleotide Variants ◽

Candida Auris ◽

Healthcare Settings ◽

Long Read ◽

Outbreak Investigations ◽

Phylogenetic Grouping

Abstract Background Candida auris (C. auris), an emerging yeast species, is often drug-resistant and has caused outbreaks in healthcare settings. Surging C. auris cases at our institution prompted whole genome sequencing (WGS) of patient and environmental specimens and comparison to local and international isolates. Methods WGS was performed on clinical and environmental isolates obtained from Northwestern Memorial Hospital (NMH) from June 2018 to December 2019. Genome sequences were compared against isolates from other institutions in the Chicagoland area obtained from a reference lab (ACL) and from the CDC. Two isolates underwent long-read sequencing on the Oxford Nanopore GridION platform to obtain closed genomes. WGS was performed on the remaining isolates with the Illumina MiSeq platform. Results Twenty isolates from NMH, five from ACL, and two from the CDC underwent WGS to yield 12.6 Mb genomes. Any two NMH isolates differed from each other by a maximum of 36 single nucleotide variants (SNV) (Figure 1). Two patients thought to be part of a transmission cluster (isolates CA06 and CA07), differed by 7 SNVs. No phylogenetic grouping between hospital systems across Chicagoland was observed. Isolates from room surfaces from a C. auris patient differed by 1-6 SNVs from each other and from 7-8 SNVs from the patient isolate. Samples taken from different body sites of another patient differed by 4-9 SNVs. Average SNV counts were lower among nosocomially acquired cases when compared to C. auris isolates present on admission (Figure 2). All NMH isolates were fluconazole sensitive, but a fluconazole resistant ACL isolate differed from a sensitive NMH isolate by only 4 SNVs. Figure 1: Phylogenetic tree of all NMH and ACL isolates with fluconazole sensitivities Figure 2: Observed pairwise SNP differences between nosocomial and POA strains Conclusion WGS of C. auris did not reveal identical isolates in any instance, even from the same patient or the patients and their environment. Generally, lower numbers of SNVs were observed for intra- versus inter-institutional isolates. More work is needed to determine the use, if any, of WGS in outbreak investigations. Disclosures All Authors: No reported disclosures

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Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing

Viruses ◽

10.3390/v12080841 ◽

2020 ◽

Vol 12 (8) ◽

pp. 841 ◽

Cited By ~ 1

Author(s):

Carole Grädel ◽

Miguel A. Terrazos Miani ◽

Christian Baumann ◽

Maria Teresa Barbani ◽

Stefan Neuenschwander ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Rna Sequencing ◽

Genome Sequencing ◽

Illumina Miseq ◽

High Viral Load ◽

Clinical Samples ◽

Whole Genome ◽

Consensus Sequences ◽

Positive Stool ◽

Stool Samples

Enteroviruses are small RNA viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. In routine diagnostic laboratories, enteroviruses are identified by PCR-based methods, often combined with partial sequencing for genotyping. In this proof-of-principle study, we assessed direct RNA sequencing (DRS) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. The approach was complemented by sequencing the corresponding viral cDNA via Illumina MiSeq sequencing. DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% and 99.6% of the most similar reference genome sequences. The identification of the enterovirus sequences in the samples was confirmed by short-read cDNA sequencing. Sequence identity between DRS and Illumina MiSeq enterovirus consensus sequences ranged between 94% and 97%. Here, we show that nanopore DRS can be used to correctly identify enterovirus genotypes from patient stool samples with high viral load and that the approach also provides rich metatranscriptomic information on sample composition for all life domains.

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