scholarly journals Real time application of whole genome sequencing for outbreak investigation – What is an achievable turnaround time?

2016 ◽  
Vol 85 (3) ◽  
pp. 277-282 ◽  
Author(s):  
Patrick McGann ◽  
Jessica L. Bunin ◽  
Erik Snesrud ◽  
Seema Singh ◽  
Rosslyn Maybank ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
Turnaround Time ◽  
Outbreak Investigation ◽  
Whole Genome ◽  
Real Time Application

scholarly journals High-precision and cost-efficient sequencing for real-time COVID-19 surveillance

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sung Yong Park ◽  
Gina Faraci ◽  
Pamela M. Ward ◽  
Jane F. Emerson ◽  
Ha Youn Lee
Keyword(s):  
Los Angeles ◽  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
High Precision ◽  
High Throughput Sequencing ◽  
Whole Genome ◽  
Sequencing Data ◽  
Public Health Response ◽  
Cost Efficient

AbstractCOVID-19 global cases have climbed to more than 33 million, with over a million total deaths, as of September, 2020. Real-time massive SARS-CoV-2 whole genome sequencing is key to tracking chains of transmission and estimating the origin of disease outbreaks. Yet no methods have simultaneously achieved high precision, simple workflow, and low cost. We developed a high-precision, cost-efficient SARS-CoV-2 whole genome sequencing platform for COVID-19 genomic surveillance, CorvGenSurv (Coronavirus Genomic Surveillance). CorvGenSurv directly amplified viral RNA from COVID-19 patients’ Nasopharyngeal/Oropharyngeal (NP/OP) swab specimens and sequenced the SARS-CoV-2 whole genome in three segments by long-read, high-throughput sequencing. Sequencing of the whole genome in three segments significantly reduced sequencing data waste, thereby preventing dropouts in genome coverage. We validated the precision of our pipeline by both control genomic RNA sequencing and Sanger sequencing. We produced near full-length whole genome sequences from individuals who were COVID-19 test positive during April to June 2020 in Los Angeles County, California, USA. These sequences were highly diverse in the G clade with nine novel amino acid mutations including NSP12-M755I and ORF8-V117F. With its readily adaptable design, CorvGenSurv grants wide access to genomic surveillance, permitting immediate public health response to sudden threats.

scholarly journals Genome Sequence ofListeria monocytogenesStrain HPB2088 (Serotype 1/2a), an Environmental Isolate Collected in Canada in 1994

2016 ◽  
Vol 4 (4) ◽  
Author(s):  
Arthur W. Pightling ◽  
Hugh Rand ◽  
Errol Strain ◽  
Franco Pagotto
Keyword(s):  
Listeria Monocytogenes ◽  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
Genome Sequence ◽  
Foodborne Pathogen ◽  
Environmental Isolate ◽  
Severe Illness ◽  
Whole Genome ◽  
Serotype 1

Listeria monocytogenesis a foodborne pathogen that causes severe illness. Thus, ongoing efforts at real-time whole-genome sequencing are of utmost importance. However, it is also important that retrospective analyses that place these data into context be performed. Here, we present the genome sequence of strain HPB2088, which was collected in 1994.

scholarly journals Real-Time Whole-Genome Sequencing for Surveillance ofListeria monocytogenes, France

2017 ◽  
Vol 23 (9) ◽  
pp. 1462-1470 ◽  
Author(s):  
Alexandra Moura ◽  
Mathieu Tourdjman ◽  
Alexandre Leclercq ◽  
Estelle Hamelin ◽  
Edith Laurent ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Oflisteria Monocytogenes

scholarly journals Real time, field-deployable whole genome sequencing of malaria parasites using nanopore technology

2020 ◽  
Author(s):  
Zahra Razook ◽  
Somya Mehra ◽  
Brittany Gilchrist ◽  
Digjaya Utama ◽  
Dulcie Lautu-Gumal ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
Error Rates ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Virulence Determinants ◽  
Malaria Parasites ◽  
Additional Information ◽  
Natural Isolates

ABSTRACTMalaria parasite genomes have been generated predominantly using short read sequencing technology which can be slow, requires advanced laboratory training and does not adequately interrogate complex genomic regions that harbour important malaria virulence determinants. The portable Oxford Nanopore Technologies MinION platform generates long reads in real time and may overcome these limitations. We present compelling evidence that Nanopore sequencing delivers valuable additional information for malaria parasites with comparable data fidelity for single nucleotide variant (SNV) calls, compared to standard Illumina whole genome sequencing. We demonstrate this through sequencing of pure Plasmodium falciparum DNA, mock infections and natural isolates. Nanopore has low error rates for haploid SNV genotyping and identifies structural variants (SVs) not detected with short reads. Nanopore genomes are directly comparable to publically available genomes and produce high quality end to end chromosome assemblies. Nanopore sequencing will expedite genomic surveillance of malaria and provide new insights into parasite genome biology.

scholarly journals Using whole-genome sequencing and a pentaplex real-time PCR to characterize third-generation cephalosporin-resistant Enterobacteriaceae from Southeast Queensland, Australia

2020 ◽  
Author(s):  
AG Stewart ◽  
EP Price ◽  
K Schabacker ◽  
M Birikmen ◽  
PNA Harris ◽  
...  
Keyword(s):  
Intensive Care Unit ◽  
Intensive Care ◽  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
Real Time Pcr ◽  
Generation Cephalosporin ◽  
Whole Genome ◽  
Third Generation ◽  
Sensitivity Testing

AbstractThird-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae represent a major threat to human health. Here, we captured 288 3GC-R Enterobacteriaceae clinical isolates from 258 patients presenting at a regional Australian hospital over a 14-month period. Alongside routine mass spectrometry speciation and antibiotic sensitivity testing, isolates were examined using a rapid (~40 min) pentaplex real-time PCR assay targeting the most common extended spectrum β-lactamases (ESBLs; CTX-M-1 and CTX-M-9 groups, plus TEM, SHV, and an internal 16S ribosomal DNA control). Additionally, AmpC CMY β-lactamase prevalence was examined using a singleplex PCR. A subset of isolates, including all 3GC-R isolates obtained from the intensive care unit, were subjected to whole-genome sequencing (WGS) to assess transmission dynamics, the presence of unidentified resistance determinants, and genotyping accuracy. Escherichia coli (80.2%) and Klebsiella pneumoniae (17.0%) were dominant, with Klebsiella oxytoca, Klebsiella aerogenes and Enterobacter cloacae infrequently identified. Ceftriaxone and cefoxitin resistance was identified in 97% and 24.5% of E. coli and K. pneumoniae isolates, respectively. Consistent with global findings in Enterobacteriaceae, the majority (98.3%) of isolates harbored at least one β-lactamase gene, with 144 (50%) encoding blaCTX-M-1 group, 92 (31.9%) blaCTX-M-9 group, 48 (16.7%) blaSHV, 133 (46.2%) blaTEM, and 34 (11.8%) blaCMY. WGS of β-lactamase negative or carbapenem-resistant isolates identified uncommon ESBLs and carbapenemases, including blaNDM and blaIMP, and confirmed all PCR-positive genotypes. No evidence of transmission among intensive care unit patients was identified. We demonstrate that our PCR assays enable the rapid and cost-effective identification of ESBLs in the hospital setting, which has important infection control and therapeutic implications.

scholarly journals AmpliCoV: Rapid Whole-Genome Sequencing Using Multiplex PCR Amplification and Real-Time Oxford Nanopore MinION Sequencing Enables Rapid Variant Identification of SARS-CoV-2

2021 ◽  
Vol 12 ◽  
Author(s):  
Annika Brinkmann ◽  
Sophie-Luisa Ulm ◽  
Steven Uddin ◽  
Sophie Förster ◽  
Dominique Seifert ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
Phylogenetic Analyses ◽  
Diagnostic Methods ◽  
Virus Concentration ◽  
Sequence Information ◽  
Whole Genome ◽  
Oxford Nanopore ◽  
A Genome

Since the emergence of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in December 2019, the scientific community has been sharing data on epidemiology, diagnostic methods, and whole-genomic sequences almost in real time. The latter have already facilitated phylogenetic analyses, transmission chain tracking, protein modeling, the identification of possible therapeutic targets, timely risk assessment, and identification of novel variants. We have established and evaluated an amplification-based approach for whole-genome sequencing of SARS-CoV-2. It can be used on the miniature-sized and field-deployable sequencing device Oxford Nanopore MinION, with sequencing library preparation time of 10 min. We show that the generation of 50,000 total reads per sample is sufficient for a near complete coverage (>90%) of the SARS-CoV-2 genome directly from patient samples even if virus concentration is low (Ct 35, corresponding to approximately 5 genome copies per reaction). For patient samples with high viral load (Ct 18–24), generation of 50,000 reads in 1–2 h was shown to be sufficient for a genome coverage of >90%. Comparison to Illumina data reveals an accuracy that suffices to identify virus mutants. AmpliCoV can be applied whenever sequence information on SARS-CoV-2 is required rapidly, for instance for the identification of circulating virus mutants.

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

2016 ◽  
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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