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 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 SureSelect targeted enrichment, a new cost effective method for the whole genome sequencing of Candidatus Liberibacter asiaticus

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Weili Cai ◽  
Schyler Nunziata ◽  
John Rascoe ◽  
Michael J. Stulberg
Keyword(s):  
Whole Genome Sequencing ◽  
Molecular Characterization ◽  
Genome Sequencing ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Genome Coverage ◽  
Metagenomic Sample ◽  
Liberibacter Asiaticus

AbstractHuanglongbing (HLB) is a worldwide deadly citrus disease caused by the phloem-limited bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) vectored by Asian citrus psyllids. In order to effectively manage this disease, it is crucial to understand the relationship among the bacterial isolates from different geographical locations. Whole genome sequencing approaches will provide more precise molecular characterization of the diversity among populations. Due to the lack of in vitro culture, obtaining the whole genome sequence of CLas is still a challenge, especially for medium to low titer samples. Hundreds of millions of sequencing reads are needed to get good coverage of CLas from an HLB positive citrus sample. In order to overcome this limitation, we present here a new method, Agilent SureSelect XT HS target enrichment, which can specifically enrich CLas from a metagenomic sample while greatly reducing cost and increasing whole genome coverage of the pathogen. In this study, the CLas genome was successfully sequenced with 99.3% genome coverage and over 72X sequencing coverage from low titer tissue samples (equivalent to 28.52 Cq using Li 16 S qPCR). More importantly, this method also effectively captures regions of diversity in the CLas genome, which provides precise molecular characterization of different strains.

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 Genomic Characterization and Phylogenetic Classification of Bovine Coronaviruses Through Whole Genome Sequence Analysis

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 183 ◽  
Author(s):  
Tohru Suzuki ◽  
Yoshihiro Otake ◽  
Satoko Uchimoto ◽  
Ayako Hasebe ◽  
Yusuke Goto
Keyword(s):  
Phylogenetic Analysis ◽  
Sequence Analysis ◽  
Molecular Characterization ◽  
Genome Sequence ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Wild Ruminant ◽  
Genome Sequence Analysis ◽  
Genomic Characterization

Bovine coronavirus (BCoV) is zoonotically transmissible among species, since BCoV-like viruses have been detected in wild ruminants and humans. BCoV causing enteric and respiratory disease is widespread in cattle farms worldwide; however, limited information is available regarding the molecular characterization of BCoV because of its large genome size, despite its significant economic impact. This study aimed to better understand the genomic characterization and evolutionary dynamics of BCoV via comparative sequence and phylogenetic analyses through whole genome sequence analysis using 67 BCoV isolates collected throughout Japan from 2006 to 2017. On comparing the genomic sequences of the 67 BCoVs, genetic variations were detected in 5 of 10 open reading frames (ORFs) in the BCoV genome. Phylogenetic analysis using whole genomes from the 67 Japanese BCoV isolates in addition to those from 16 reference BCoV strains, revealed the existence of two major genotypes (classical and US wild ruminant genotypes). All Japanese BCoV isolates originated from the US wild ruminant genotype, and they tended to form the same clusters based on the year and farm of collection, not the disease type. Phylogenetic trees on hemagglutinin-esterase protein (HE), spike glycoprotein (S), nucleocapsid protein (N) genes and ORF1 revealed clusters similar to that on whole genome, suggesting that the evolution of BCoVs may be closely associated with variations in these genes. Furthermore, phylogenetic analysis of BCoV S genes including those of European and Asian BCoVs and human enteric coronavirus along with the Japanese BCoVs revealed that BCoVs differentiated into two major types (European and American types). Moreover, the European and American types were divided into eleven and three genotypes, respectively. Our analysis also demonstrated that BCoVs with different genotypes periodically emerged and predominantly circulated within the country. These findings provide useful information to elucidate the detailed molecular characterization of BCoVs, which have spread worldwide. Further genomic analyses of BCoV are essential to deepen the understanding of the evolution of this virus.

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 Full Genomic Characterization of a Lentogenic Newcastle Disease Virus Isolated from Farm-Reared Ostriches (Struthio camelus) in Northwest China

2017 ◽  
Vol 5 (6) ◽  
Author(s):  
Shanhui Ren ◽  
Chongyang Wang ◽  
Xiaolong Gao ◽  
Xue Zhang ◽  
Xiangwei Wang ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Northwest China ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Struthio Camelus ◽  
Genomic Characterization

ABSTRACT To our knowledge, our study is the first to report the whole-genome sequence of an ostrich-origin Newcastle disease virus (NDV) isolate, abbreviated as Ostrich/SX-01/06. Phylogenetic analysis revealed that this isolate belongs to the subgenotype c in class II. The identification of the complete genome will provide useful information regarding ostrich diseases, especially NDV.

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.

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