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 A novel genotype of Hantaan orthohantavirus harbored by Apodemus agrarius chejuensis as a potential etiologic agent of hemorrhagic fever with renal syndrome in Republic of Korea

2021 ◽  
Vol 15 (5) ◽  
pp. e0009400
Author(s):  
Kyungmin Park ◽  
Won-Keun Kim ◽  
Seung-Ho Lee ◽  
Jongwoo Kim ◽  
Jingyeong Lee ◽  
...  
Keyword(s):  
Phylogenetic Analyses ◽  
Hemorrhagic Fever ◽  
Etiologic Agent ◽  
Jeju Island ◽  
Etiological Agent ◽  
Republic Of Korea ◽  
Hantaan Virus ◽  
Genomic Sequences ◽  
Mortality And Morbidity ◽  
Apodemus Agrarius

Background Orthohantaviruses, causing hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome, pose a significant public health threat worldwide. Despite the significant mortality and morbidity, effective antiviral therapeutics or vaccines for orthohantavirus infections are currently unavailable. This study aimed to investigate the prevalence of HFRS-associated orthohantaviruses and identify the etiological agent of orthohantavirus outbreaks in southern Republic of Korea (ROK). Methodology/Principal findings We collected small mammals on Jeju Island during 2018–2020. We detected the Hantaan virus (HTNV)-specific antibodies and RNA using an indirect immunofluorescence assay test and reverse transcription-polymerase chain reaction on Apodemus agrarius chejuensis (A. chejuensis). The prevalence of anti-HTNV antibodies among rodents was 14.1%. A total of six seropositive mice harbored HTNV RNA. The amplicon-based next-generation sequencing provided nearly full-length tripartite genomic sequences of six HTNV harbored by A. chejuensis. Phylogenetic and tanglegram analyses were conducted for inferring evolutionary relationships between orthohantaviruses with their reservoir hosts. Phylogenetic analyses identified a novel distinct HTNV genotype. The detected HTNV genomic sequences were phylogenetically related to a viral sequence derived from HFRS patient in southern ROK. Tanglegram analysis demonstrated the segregation of HTNV genotypes corresponding to Apodemus spp. divergence. Conclusions/Significance Our results suggest that A. chejuensis-borne HTNV may be a potential etiological agent of HFRS in southern ROK. Ancestral HTNV may infect A. chejuensis prior to geological isolation between the Korean peninsula and Jeju Island, supporting the co-evolution of orthohantaviruses and rodents. This study arises awareness among physicians for HFRS outbreaks in southern ROK.

Genomic sequences for fungi.

2021 ◽  
pp. 231-254
Author(s):  
Riccardo Baroncelli ◽  
Giovanni Cafà
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Fungal Genome ◽  
Basic Knowledge ◽  
Genomic Sequences ◽  
Whole Genome ◽  
Fungal Genomics

Abstract This chapter aims to give an overview of the basic knowledge, understanding and perspectives in fungal genomics. It is likely that fungal genome sequencing will soon become simpler and cheaper, and allow most research laboratories to undertake in-house, whole genome sequencing on a regular basis, as sequencers will be accessible to most laboratories. Nonetheless, most of the innovation in the next decade will be driven by theories in innovative perspectives and fields of investigation, rather than in novel technical approaches.

scholarly journals Comparison of targeted next-generation sequencing for whole-genome sequencing of Hantaan orthohantavirus in Apodemus agrarius lung tissues

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jin Sun No ◽  
Won-Keun Kim ◽  
Seungchan Cho ◽  
Seung-Ho Lee ◽  
Jeong-Ah Kim ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Clinical Samples ◽  
Whole Genome ◽  
Target Enrichment ◽  
Next Generation ◽  
Apodemus Agrarius ◽  
Target Capture ◽  
Generation Sequencing

Abstract Orthohantaviruses, negative-sense single-strand tripartite RNA viruses, are a global public health threat. In humans, orthohantavirus infection causes hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. Whole-genome sequencing of the virus helps in identification and characterization of emerging or re-emerging viruses. Next-generation sequencing (NGS) is a potent method to sequence the viral genome, using molecular enrichment methods, from clinical specimens containing low virus titers. Hence, a comparative study on the target enrichment NGS methods is required for whole-genome sequencing of orthohantavirus in clinical samples. In this study, we used the sequence-independent, single-primer amplification, target capture, and amplicon NGS for whole-genome sequencing of Hantaan orthohantavirus (HTNV) from rodent specimens. We analyzed the coverage of the HTNV genome based on the viral RNA copy number, which is quantified by real-time quantitative PCR. Target capture and amplicon NGS demonstrated a high coverage rate of HTNV in Apodemus agrarius lung tissues containing up to 103–104 copies/μL of HTNV RNA. Furthermore, the amplicon NGS showed a 10-fold (102 copies/μL) higher sensitivity than the target capture NGS. This report provides useful insights into target enrichment NGS for whole-genome sequencing of orthohantaviruses without cultivating the viruses.

scholarly journals Identification and Whole-Genome Sequencing of Four Enterovirus D68 Strains in Southern China in Late 2015

2016 ◽  
Vol 4 (5) ◽  
Author(s):  
Long Chen ◽  
Lei Shi ◽  
Hong Yang ◽  
Da-Yong Gu ◽  
Jun Meng ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Phylogenetic Relationship ◽  
Genome Sequencing ◽  
Southern China ◽  
Northern China ◽  
Genomic Sequences ◽  
Whole Genome ◽  
Influenza Like Illness ◽  
Viral Genomes ◽  
Enterovirus D68

Four enterovirus D68 (EV-D68) strains from four children with influenza-like illness were identified in Shenzhen, southern China, in late 2015. Here, we announce the availability of these viral genomes in GenBank. The genomic sequences of these EV-D68 strains showed the closest phylogenetic relationship to strains from northern China.

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 Identifying transposon insertions in bacterial genomes through nanopore sequencing

2019 ◽  
Author(s):  
David A. Baltrus ◽  
John Medlen ◽  
Meara Clark
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Genetic Screens ◽  
Bacterial Genomes ◽  
Sequence Capture ◽  
Transposon Insertion ◽  
Forward Genetic Screens ◽  
Transposon Insertions

AbstractTransposon mutagenesis is a widely used tool for carrying out forward genetic screens across systems, but in some cases it can be difficult to identify transposon insertion points after successful phenotypic screens. As an alternative to traditional methods, we report on the efficacy of using an Oxford Nanopore’s MinION to identify transposon insertions through whole genome sequencing. We also report experiments using CRISPR-Cas to selectively target regions of the genome where a transposon has integrated. Our experiments provide a framework for understanding the efficiency of such techniques for carrying out forward genetic screens and point towards the ability to use CRISPR-based sequence capture to identify the insertion of particular regions of DNA across all genomes, which may enable Tn-Seq experiments using Nanopore based sequencing.

scholarly journals Evaluating an Upper Respiratory Disease Panel on the Portable MinION Sequencer

2018 ◽  
Author(s):  
Wanda J. Lyon ◽  
Zachary K. Smith ◽  
Brian Grier ◽  
James Baldwin ◽  
Clarise R. Starr
Keyword(s):  
Whole Genome Sequencing ◽  
Respiratory Disease ◽  
Genome Sequencing ◽  
Error Rate ◽  
Whole Genome Amplification ◽  
Targeted Sequencing ◽  
Whole Genome ◽  
Genome Amplification ◽  
Upper Respiratory ◽  
Nanopore Sequencer

AbstractThe MinION was used to evaluate upper respiratory disease infections using both whole genome amplification (WGA), targeted sequencing, and was found to have tremendous potential for field use. The MinION nanopore sequencer was been released to community testers for evaluation using a variety of sequencing applications. The MinION was used to evaluate upper respiratory disease infections using both whole genome amplification and targeted sequencing, and was found to have tremendous potential for field use. In this study, we tested the ability of the MinION nanopore sequencer to accurately identify and differentiate clinical bacterial and viral samples via targeted sequencing and whole genome sequencing. The current nanopore technology has limitations with respect to error rate but has steadily improved with development of new flow cells and kits. Upper respiratory disease organisms were successfully identified and differentiated down to the strain level with 87-98% alignment to our reference genome database. The ability to differentiate strains by amplicon and whole genome sequencing on the MinION was accomplished despite the observed average per 100-base error rate averaged 1.2E-01. This study offers evidence of the utility of sequencing to identify and differentiate both viral and bacterial species present within clinical samples.

scholarly journals Viral loads and profile of the patients infected with SARS-CoV-2 Delta, Alpha or R.1 variants in Tokyo

2021 ◽  
Author(s):  
Chihiro Tani-Sassa ◽  
Yumi Iwasaki ◽  
Naoya Ichimura ◽  
Katsutoshi Nagano ◽  
Yuna Takatsuki ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Copy Number ◽  
Whole Genome ◽  
Elderly Individuals ◽  
Viral Loads ◽  
Copy Numbers ◽  
Significant Difference ◽  
Rapid Spread ◽  
Alpha Variant

The rapid spread of the Delta variant of SARS-CoV-2 became a serious concern worldwide in summer 2021. We examined the copy number and variant types of all SARS-CoV-2-positive patients who visited our hospital from February to August 2021 using PCR tests. Whole genome sequencing was performed for some samples. The R.1 variant (B.1.1.316) was responsible for most infections in March, replacing the previous variant (B.1.1.214); the Alpha (B.1.1.7) variant caused most infections in April and May; and the Delta variant (B.1.617.2) was the most prevalent in July and August. There was no significant difference in copy numbers among the previous variant cases (n=29, median 3.0x104 copies/μL), R.1 variant cases (n=28, 2.1x105 copies/μL), Alpha variant cases (n=125, 4.1x105 copies/μL), and Delta variant cases (n=106, 2.4x105 copies/μL). Patients with Delta variant infection were significantly younger than those infected with R.1 and the previous variants, possibly because many elderly individuals in Tokyo were vaccinated between May and August. There was no significant difference in mortality among the four groups. Our results suggest that the increased infectivity of Delta variant may be caused by factors other than the higher viral loads. Clarifying these factors is important to control the spread of Delta variant infection.

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