scholarly journals Rapid genomic characterization of SARS-CoV-2 viruses from clinical specimens using nanopore sequencing

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jun Li ◽  
Haoqiu Wang ◽  
Lingfeng Mao ◽  
Hua Yu ◽  
Xinfen Yu ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Clinical Samples ◽  
Nanopore Sequencing ◽  
The Novel ◽  
Multiple Sources ◽  
Clinical Specimens ◽  
Whole Genomes ◽  
Genomic Characterization ◽  
Nanopore Sequencer

Abstract The novel SARS-CoV-2 outbreak has swiftly spread worldwide. The rapid genome sequencing of SARS-CoV-2 strains has become a helpful tool for better understanding the genomic characteristics and origin of the virus. To obtain virus whole-genome sequences directly from clinical specimens, we performed nanopore sequencing using a modified ARTIC protocol in a portable nanopore sequencer and validated a routine 8-h workflow and a 5-h rapid pipeline. We conducted some optimization to improve the genome sequencing workflow. The sensitivity of the workflow was also tested by serially diluting RNA from clinical samples. The optimized pipeline was finally applied to obtain the whole genomes of 29 clinical specimens collected in Hangzhou from January to March 2020. In the 29 obtained complete genomes of SARS-CoV-2, 33 variations were identified and analyzed. The genomic variations and phylogenetic analysis hinted at multiple sources and different transmission patterns during the COVID-19 epidemic in Hangzhou, China. In conclusion, the genomic characteristics and origin of the virus can be quickly determined by nanopore sequencing following our workflows.

scholarly journals Rapid Genomic Characterization of SARS-CoV-2 Viruses From Clinical Specimens Using Nanopore Sequencing

2020 ◽  
Author(s):  
Jun Li ◽  
Haoqiu Wang ◽  
Lingfeng Mao ◽  
Hua Yu ◽  
Xinfen Yu ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Whole Genome Sequence ◽  
Clinical Samples ◽  
Nanopore Sequencing ◽  
The Novel ◽  
Multiple Sources ◽  
Clinical Specimens ◽  
Whole Genomes ◽  
Genomic Characterization

Abstract The outbreak of the novel SARS-CoV-2 has swiftly spread worldwide. Rapid genome sequencing of the SARS-CoV-2 strains has become a helpful tool for better understanding of virus genomic characteristics and the origin. To obtain the virus whole-genome sequence directly from the clinical specimens, we performed the nanopore sequencing using a modified ARTIC protocol on portable nanopore sequencer, and validated the routine 8 hours workflow and 5 hours rapid pipeline. We had made some optimizations to improve the genome sequencing workflow. The sensitivity of the workflow was also tested by serially diluting RNA from clinical samples. The optimized pipeline was finally applied to obtain the whole genomes from 17 clinical specimens in Hangzhou from January 2020 to March 2020. In the obtained 17 complete genomes of SARS-CoV-2, 12 variations were found and analyzed. The genomic variations and phylogenetic analysis hinted the multiple sources and different transmission pattern during the COVID-19 epidemic in Hangzhou, China. In conclusion, the genomic characteristics and the origin of the virus could be quickly determined by nanopore sequencing following our workflows.

scholarly journals Multiplex PCR-Based Nanopore Sequencing and Epidemiological Surveillance of Hantaan orthohantavirus in Apodemus agrarius, Republic of Korea

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 847
Author(s):  
Kyungmin Park ◽  
Seung-Ho Lee ◽  
Jongwoo Kim ◽  
Jingyeong Lee ◽  
Geum-Young Lee ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Republic Of Korea ◽  
Hantaan Virus ◽  
Genomic Sequences ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Apodemus Agrarius ◽  
Copy Numbers ◽  
Nanopore Sequencer

Whole-genome sequencing of infectious agents enables the identification and characterization of emerging viruses. The MinION device is a portable sequencer that allows real-time sequencing in fields or hospitals. Hantaan orthohantavirus (Hantaan virus, HTNV), harbored by Apodemus agrarius, causes hemorrhagic fever with renal syndrome (HFRS) and poses a critical public health threat worldwide. In this study, we aimed to evaluate the feasibility of using nanopore sequencing for whole-genome sequencing of HTNV from samples having different viral copy numbers. Amplicon-based next-generation sequencing was performed in A. agrarius lung tissues collected from the Republic of Korea. Genomic sequences of HTNV were analyzed based on the viral RNA copy numbers. Amplicon-based nanopore sequencing provided nearly full-length genomic sequences of HTNV and showed sufficient read depth for phylogenetic analysis after 8 h of sequencing. The average identity of the HTNV genome sequences for the nanopore sequencer compared to those of generated from Illumina MiSeq revealed 99.8% (L and M segments) and 99.7% (S segment) identities, respectively. This study highlights the potential of the portable nanopore sequencer for rapid generation of accurate genomic sequences of HTNV for quicker decision making in point-of-care testing of HFRS patients during a hantavirus outbreak.

scholarly journals Optimizing DNA extraction methods for Nanopore sequencing of Neisseria gonorrhoeae direct from urine samples

2019 ◽  
Author(s):  
Teresa L. Street ◽  
Leanne Barker ◽  
Nicholas D. Sanderson ◽  
James Kavanagh ◽  
Sarah Hoosdally ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Reference Genome ◽  
Extraction Methods ◽  
Urine Samples ◽  
Clinical Samples ◽  
Nanopore Sequencing ◽  
Human Dna ◽  
Whole Genomes ◽  
Dna Extraction Methods ◽  
Multiple Antibiotics

AbstractBackgroundEmpirical gonorrhoea treatment at initial diagnosis reduces onward transmission. However, increasing resistance to multiple antibiotics may necessitate waiting for culture-based diagnostics to select an effective treatment. There is a need for same-day culture-free diagnostics that identify infection and detect antimicrobial resistance.MethodsWe investigated if Nanopore sequencing can detect sufficient N. gonorrhoeae DNA to reconstruct whole genomes directly from urine samples. We used N. gonorrhoeae spiked urine samples and samples from gonorrhoea infections to determine optimal DNA extraction methods that maximize the amount of N. gonorrhoeae DNA sequenced whilst minimizing contaminating host DNA.ResultsIn simulated infections the Qiagen UCP Pathogen Mini kit provided the highest ratio N. gonorrhoeae to human DNA and the most consistent results. Depletion of human DNA with saponin increased N. gonorrhoeae yields in simulated infections, but decreased yields in clinical samples. In ten urine samples from men with symptomatic urethral gonorrhoea, ≥87% coverage of an N. gonorrhoeae reference genome was achieved in all samples, with ≥92% coverage breath at ≥10-fold depth in 7 (70%) samples. In simulated infections if ≥104 CFU/ml of N. gonorrhoeae was present, sequencing of the large majority of the genome was frequently achieved. N. gonorrhoeae could also be detected from urine in cobas PCR Media tubes and from urethral swabs, and in the presence of simulated Chlamydia co-infection.ConclusionUsing Nanopore sequencing of urine samples from men with urethral gonorrhoea sufficient data can be obtained to reconstruct whole genomes in the majority of samples without the need for culture.

scholarly journals Complete Genome Sequence of an Avian Paramyxovirus Representative of Putative New Serotype 13

2016 ◽  
Vol 4 (4) ◽  
Author(s):  
Iryna Goraichuk ◽  
Poonam Sharma ◽  
Borys Stegniy ◽  
Denys Muzyka ◽  
Mary J. Pantin-Jackwood ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
The Novel ◽  
Avian Paramyxovirus ◽  
Genomic Characterization

Here, we report the complete genome sequence of a virus of a putative new serotype of avian paramyxovirus (APMV). The virus was isolated from a white-fronted goose in Ukraine in 2011 and designated white-fronted goose/Ukraine/Askania-Nova/48-15-02/2011. The genomic characterization of the isolate suggests that it represents the novel avian paramyxovirus group APMV 13.

scholarly journals Rapid Whole-Genome Sequencing for Detection and Characterization of Microorganisms Directly from Clinical Samples

2014 ◽  
Vol 52 (8) ◽  
pp. 3136-3136 ◽  
Author(s):  
H. Hasman ◽  
D. Saputra ◽  
T. Sicheritz-Ponten ◽  
O. Lund ◽  
C. A. Svendsen ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Clinical Samples ◽  
Whole Genome

scholarly journals Characterization of Genetically Modified Microorganisms Using Short- and Long-Read Whole-Genome Sequencing Reveals Contaminations of Related Origin in Multiple Commercial Food Enzyme Products

Foods ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2637
Author(s):  
Jolien D’aes ◽  
Marie-Alice Fraiture ◽  
Bert Bogaerts ◽  
Sigrid C. J. De Keersmaecker ◽  
Nancy H. C. Roosens ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genetically Modified ◽  
Added Value ◽  
Phylogenomic Analysis ◽  
Whole Genome ◽  
Antimicrobial Resistance Genes ◽  
Long Read ◽  
Genomic Characterization

Despite their presence being unauthorized on the European market, contaminations with genetically modified (GM) microorganisms have repeatedly been reported in diverse commercial microbial fermentation produce types. Several of these contaminations are related to a GM Bacillus velezensis used to synthesize a food enzyme protease, for which genomic characterization remains currently incomplete, and it is unknown whether these contaminations have a common origin. In this study, GM B. velezensis isolates from multiple food enzyme products were characterized by short- and long-read whole-genome sequencing (WGS), demonstrating that they harbor a free recombinant pUB110-derived plasmid carrying antimicrobial resistance genes. Additionally, single-nucleotide polymorphism (SNP) and whole-genome based comparative analyses showed that the isolates likely originate from the same parental GM strain. This study highlights the added value of a hybrid WGS approach for accurate genomic characterization of GMM (e.g., genomic location of the transgenic construct), and of SNP-based phylogenomic analysis for source-tracking of GMM.

scholarly journals Complete Genome Sequences for Two Strains of a Novel Fastidious, Partially Acid-Fast, Gram-Positive Corynebacterineae Bacterium, Derived from Human Clinical Samples

2015 ◽  
Vol 3 (6) ◽  
Author(s):  
Ainsley C. Nicholson ◽  
Melissa Bell ◽  
Ben W. Humrighouse ◽  
John R. McQuiston
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Clinical Relevance ◽  
Complete Genome ◽  
Growth Characteristics ◽  
Clinical Samples ◽  
Whole Genome ◽  
The Novel ◽  
Genome Sequences ◽  
Gram Positive

Here we report the complete genome sequences of two strains of the novel fastidious, partially acid-fast, Gram-positive bacillus “ Lawsonella clevelandensis ” (proposed). Their clinical relevance and unusual growth characteristics make them intriguing candidates for whole-genome sequencing.

scholarly journals Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing

2021 ◽  
Vol 6 ◽  
pp. 241
Author(s):  
Ingra M. Claro ◽  
Mariana S. Ramundo ◽  
Thais M. Coletti ◽  
Camila A. M. da Silva ◽  
Ian N. Valenca ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Yellow Fever Virus ◽  
Rna Virus ◽  
Clinical Sample ◽  
Viral Metagenomics ◽  
Health Concern ◽  
Clinical Samples ◽  
Nanopore Sequencing ◽  
Emerging Viruses ◽  
Genome Coverage

Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks. SMART (Switching Mechanism at the 5′ end of RNA Template) is a popular method for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, ‘SMART-9N’, and a version compatible with barcoded PCR primers available from Oxford Nanopore Technologies, ‘Rapid SMART-9N’, for the detection, characterization, and whole-genome sequencing of RNA viruses. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6e00 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method. This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work.

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