scholarly journals Adaptation of Oxford Nanopore technology for hepatitis C whole genome sequencing and identification of within-host viral variants

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Nasir Riaz ◽  
Preston Leung ◽  
Kirston Barton ◽  
Martin A. Smith ◽  
Shaun Carswell ◽  
...  
Keyword(s):  
Cost Effectiveness ◽  
Hepatitis C ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Reference Sequence ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Accurate Identification ◽  
Low Frequencies ◽  
Oxford Nanopore

Abstract Background Hepatitis C (HCV) and many other RNA viruses exist as rapidly mutating quasi-species populations in a single infected host. High throughput characterization of full genome, within-host variants is still not possible despite advances in next generation sequencing. This limitation constrains viral genomic studies that depend on accurate identification of hemi-genome or whole genome, within-host variants, especially those occurring at low frequencies. With the advent of third generation long read sequencing technologies, including Oxford Nanopore Technology (ONT) and PacBio platforms, this problem is potentially surmountable. ONT is particularly attractive in this regard due to the portable nature of the MinION sequencer, which makes real-time sequencing in remote and resource-limited locations possible. However, this technology (termed here ‘nanopore sequencing’) has a comparatively high technical error rate. The present study aimed to assess the utility, accuracy and cost-effectiveness of nanopore sequencing for HCV genomes. We also introduce a new bioinformatics tool (Nano-Q) to differentiate within-host variants from nanopore sequencing. Results The Nanopore platform, when the coverage exceeded 300 reads, generated comparable consensus sequences to Illumina sequencing. Using HCV Envelope plasmids (~ 1800 nt) mixed in known proportions, the capacity of nanopore sequencing to reliably identify variants with an abundance as low as 0.1% was demonstrated, provided the autologous reference sequence was available to identify the matching reads. Successful pooling and nanopore sequencing of 52 samples from patients with HCV infection demonstrated its cost effectiveness (AUD$ 43 per sample with nanopore sequencing versus $100 with paired-end short read technology). The Nano-Q tool successfully separated between-host sequences, including those from the same subtype, by bulk sorting and phylogenetic clustering without an autologous reference sequence (using only a subtype-specific generic reference). The pipeline also identified within-host viral variants and their abundance when the parameters were appropriately adjusted. Conclusion Cost effective HCV whole genome sequencing and within-host variant identification without haplotype reconstruction are potential advantages of nanopore sequencing.

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.

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

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

scholarly journals Using molecular testing and whole-genome sequencing for tuberculosis diagnosis in a low-burden setting: a cost-effectiveness analysis using transmission-dynamic modelling

Thorax ◽  
2021 ◽  
Vol 76 (3) ◽  
pp. 281-291 ◽  
Author(s):  
Tendai Mugwagwa ◽  
Ibrahim Abubakar ◽  
Peter J White
Keyword(s):  
Cost Effectiveness ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Drug Sensitivity ◽  
Cost Effective ◽  
Molecular Testing ◽  
Whole Genome ◽  
Transmission Dynamic ◽  
Sensitivity Testing ◽  
Combined Use

BackgroundDespite progress in TB control in low-burden countries like England and Wales, there are still diagnostic delays. Molecular testing and/or whole-genome sequencing (WGS) provide more rapid diagnosis but their cost-effectiveness is relatively unexplored in low-burden settings.MethodsAn integrated transmission-dynamic health economic model is used to assess the cost-effectiveness of using WGS to replace culture-based drug-sensitivity testing, versus using molecular testing versus combined use of WGS and molecular testing, for routine TB diagnosis. The model accounts for the effects of faster appropriate treatment in reducing transmission, benefiting health and reducing future treatment costs. Cost-effectiveness is assessed using incremental net benefit (INB) over a 10-year horizon with a quality-adjusted life-year valued at £20 000, and discounting at 3.5% per year.ResultsWGS shortens the time to drug sensitivity testing and treatment modification where necessary, reducing treatment and hospitalisation costs, with an INB of £7.1 million. Molecular testing shortens the time to TB diagnosis and treatment. Initially, this causes an increase in annual costs of treatment, but averting transmissions and future active TB disease subsequently, resulting in cost savings and health benefits to achieve an INB of £8.6 million (GeneXpert MTB/RIF) or £11.1 million (Xpert-Ultra). Combined use of Xpert-Ultra and WGS is the optimal strategy we consider, with an INB of £16.5 million.ConclusionRoutine use of WGS or molecular testing is cost-effective in a low-burden setting, and combined use is the most cost-effective option. Adoption of these technologies can help low-burden countries meet the WHO End TB Strategy milestones, particularly the UK, which still has relatively high TB rates.

scholarly journals Draft Genome Sequences of Six Strains Isolated from the InSight Spacecraft and Associated Surfaces Using Oxford Nanopore- and Illumina-Based Sequencing

2020 ◽  
Vol 9 (21) ◽  
Author(s):  
Daniel L. Vera ◽  
Arman Seuylemezian ◽  
Kyle S. Landry ◽  
Ryan Hendrickson
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Draft Genome ◽  
Whole Genome ◽  
Genome Sequences ◽  
Planetary Protection ◽  
Functional Capabilities ◽  
Oxford Nanopore ◽  
Associated Surfaces ◽  
Insight Mission

ABSTRACT Whole-genome sequencing and annotation have allowed planetary protection engineers to assess the functional capabilities of microorganisms isolated from spacecraft hardware and associated surfaces. Here, we report draft genomes of six strains isolated from the InSight mission, determined using Oxford Nanopore- and Illumina-based sequencing.

scholarly journals Draft Genome Sequences of 64 Type Strains of 50 Species and 25 Subspecies of the Genus Staphylococcus Rosenbach 1884

2019 ◽  
Vol 8 (17) ◽  
Author(s):  
Kevin Cole ◽  
Dona Foster ◽  
Julie E. Russell ◽  
Tanya Golubchik ◽  
Martin Llewelyn ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Draft Genome ◽  
Whole Genome ◽  
Genome Sequences ◽  
Accurate Identification ◽  
Access To Data ◽  
Type Strains

Members of the genus Staphylococcus have been isolated from humans, animals, and the environment. Accurate identification with whole-genome sequencing requires access to data derived from type strains.

scholarly journals Whole-Genome Sequencing of a Human Clinical Isolate of the Novel Species Klebsiella quasivariicola sp. nov

2017 ◽  
Vol 5 (42) ◽  
Author(s):  
S. Wesley Long ◽  
Sarah E. Linson ◽  
Matthew Ojeda Saavedra ◽  
Concepcion Cantu ◽  
James J. Davis ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Clinical Isolate ◽  
Genome Sequencing ◽  
Novel Species ◽  
Whole Genome ◽  
The Novel ◽  
Short Read ◽  
Oxford Nanopore ◽  
Long Read

ABSTRACT In a study of 1,777 Klebsiella strains, we discovered KPN1705, which was distinct from all recognized Klebsiella spp. We closed the genome of strain KPN1705 using a hybrid of Illumina short-read and Oxford Nanopore long-read technologies. For this novel species, we propose the name Klebsiella quasivariicola sp. nov.

scholarly journals Mycobacterial DNA Extraction for Whole-Genome Sequencing from Early Positive Liquid (MGIT) Cultures

2015 ◽  
Vol 53 (4) ◽  
pp. 1137-1143 ◽  
Author(s):  
Antonina A. Votintseva ◽  
Louise J. Pankhurst ◽  
Luke W. Anson ◽  
Marcus R. Morgan ◽  
Deborah Gascoyne-Binzi ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Solid Phase ◽  
Clinical Samples ◽  
Reference Laboratory ◽  
Whole Genome ◽  
Mycobacterial Species ◽  
Accurate Identification ◽  
Content Type

We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ≥0.2 ng/μl of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with <5% of reads derived from human or nasopharyngeal flora for 88% and 91% of samples, respectively. A total of 59 (98%) of 60 samples that were identified by the national mycobacterial reference laboratory (NMRL) asMycobacterium tuberculosiswere successfully mapped to the H37Rv reference, with >90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools.

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 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

2018 ◽  
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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