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.

scholarly journals Assessment of metagenomic Nanopore and Illumina sequencing for recovering whole genome sequences of chikungunya and dengue viruses directly from clinical samples

2018 ◽  
Vol 23 (50) ◽  
Author(s):  
Liana E. Kafetzopoulou ◽  
Kyriakos Efthymiadis ◽  
Kuiama Lewandowski ◽  
Ant Crook ◽  
Dan Carter ◽  
...  
Keyword(s):  
Illumina Miseq ◽  
Clinical Samples ◽  
Whole Genome ◽  
Metagenomic Sequencing ◽  
Front Line ◽  
Genome Sequences ◽  
Dengue Viruses ◽  
Virus Identification ◽  
Reverse Transcription Pcr ◽  
Oxford Nanopore

Background The 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. Aim To investigate the feasibility of metagenomic sequencing for recovering whole genome sequences of chikungunya and dengue viruses from clinical samples. Methods We performed metagenomic sequencing using both the Illumina MiSeq and the portable Oxford Nanopore MinION on clinical samples which were real-time reverse transcription-PCR (qRT-PCR) positive for chikungunya (CHIKV) or dengue virus (DENV), two of the most important arboviruses. A total of 26 samples with a range of representative clinical Ct values were included in the study. Results Direct metagenomic sequencing of nucleic acid extracts from serum or plasma without viral enrichment allowed for virus identification, subtype determination and elucidated complete or near-complete genomes adequate for phylogenetic analysis. One PCR-positive CHIKV sample was also found to be coinfected with DENV. Conclusions This work demonstrates that metagenomic whole genome sequencing is feasible for the majority of CHIKV and DENV PCR-positive patient serum or plasma samples. Additionally, it explores the use of Nanopore metagenomic sequencing for DENV and CHIKV, which can likely be applied to other RNA viruses, highlighting the applicability of this approach to front-line public health and potential portable applications using the MinION.

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 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 Whole-Genome Sequencing of Salmonella enterica Serovar Infantis and Kentucky Isolates Obtained from Layer Poultry Farms in Ecuador

2020 ◽  
Vol 9 (13) ◽  
Author(s):  
William Calero-Cáceres ◽  
Joyce Villacís ◽  
Maria Ishida ◽  
Elton Burnett ◽  
Christian Vinueza-Burgos
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Salmonella Enterica ◽  
Egg Production ◽  
Illumina Miseq ◽  
Whole Genome ◽  
Genome Sequences ◽  
Content Type ◽  
Poultry Farms

Five strains of Salmonella enterica subsp. enterica serovar Infantis and two strains of S. enterica subsp. enterica serovar Kentucky isolated in 2017 from Ecuadorian layer poultry farms were sequenced using Illumina MiSeq technology. These isolates were collected on layer farms in central Ecuador, one of the most important areas of egg production in the country. The genome sequences of these isolates show valuable information for surveillance purposes.

scholarly journals Identification and Whole-Genome Sequencing of a Monkeypox Virus Strain Isolated in Israel

2020 ◽  
Vol 9 (10) ◽  
Author(s):  
Inbar Cohen-Gihon ◽  
Ofir Israeli ◽  
Ohad Shifman ◽  
Noam Erez ◽  
Sharon Melamed ◽  
...  
Keyword(s):  
West Africa ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Virus Strain ◽  
Illumina Miseq ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Monkeypox Virus ◽  
Oxford Nanopore ◽  
Oxford Nanopore Technologies

We report the whole-genome sequence of a monkeypox virus strain isolated in Israel. The strain was isolated in 2018 from a patient travelling back from West Africa. The virus was fully sequenced on the Illumina MiSeq and Oxford Nanopore Technologies MinION platforms.

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 Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 841 ◽  
Author(s):  
Carole Grädel ◽  
Miguel A. Terrazos Miani ◽  
Christian Baumann ◽  
Maria Teresa Barbani ◽  
Stefan Neuenschwander ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Rna Sequencing ◽  
Genome Sequencing ◽  
Illumina Miseq ◽  
High Viral Load ◽  
Clinical Samples ◽  
Whole Genome ◽  
Consensus Sequences ◽  
Positive Stool ◽  
Stool Samples

Enteroviruses are small RNA viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. In routine diagnostic laboratories, enteroviruses are identified by PCR-based methods, often combined with partial sequencing for genotyping. In this proof-of-principle study, we assessed direct RNA sequencing (DRS) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. The approach was complemented by sequencing the corresponding viral cDNA via Illumina MiSeq sequencing. DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% and 99.6% of the most similar reference genome sequences. The identification of the enterovirus sequences in the samples was confirmed by short-read cDNA sequencing. Sequence identity between DRS and Illumina MiSeq enterovirus consensus sequences ranged between 94% and 97%. Here, we show that nanopore DRS can be used to correctly identify enterovirus genotypes from patient stool samples with high viral load and that the approach also provides rich metatranscriptomic information on sample composition for all life domains.

scholarly journals DNA Thermo-Protection Facilitates Whole Genome Sequencing of Mycobacteria Direct from Clinical Samples by the Nanopore Platform

2020 ◽  
Author(s):  
Sophie George ◽  
Yifei Xu ◽  
Gillian Rodger ◽  
Marcus Morgan ◽  
Nicholas D. Sanderson ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Low Cost ◽  
Clinical Samples ◽  
Heat Inactivation ◽  
Whole Genome ◽  
Genome Coverage ◽  
Human Dna ◽  
Oxford Nanopore ◽  
Oxford Nanopore Technologies

ABSTRACTMycobacterium tuberculosis (MTB) is the leading cause of death from bacterial infection. Improved rapid diagnosis and antimicrobial resistance determination, such as by whole genome sequencing, are required. Our aim was to develop a simple, low-cost method of preparing DNA for Oxford Nanopore Technologies (ONT) sequencing direct from MTB positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat-inactivation (99°C/30min) and enrichment for Mycobacteria DNA was achieved using an equal volume of thermo-protection buffer (4M KCl, 0.05M HEPES buffer pH7.5, 0.1% DTT). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermo-degradation, which renders it a poor template for sequencing. Initial validation employed Mycobacteria DNA (extracted or intracellular). Next, mock clinical samples (infection-negative human sputum spiked 0-105 BCG cells/ml) underwent liquefaction in thermo-protection buffer and heat-inactivation. DNA was extracted and sequenced. Human DNA degraded faster than Mycobacteria DNA, resulting in target enrichment. Four replicate experiments each demonstrated detection at 101 BCG cells/ml, with 31-59 MTB complex reads. Maximal genome coverage (>97% at 5x-depth) was achieved at 104 BCG cells/ml; >91% coverage (1x depth) at 103 BCG cells/ml. Final validation employed MTB positive clinical samples (n=20), revealed initial sample volumes ≥1ml typically yielded higher mean depth of MTB genome coverage, the overall range 0.55-81.02. A mean depth of 3 gave >96% one-fold TB genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved >99% five-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of MTB genomes was facilitated by a low cost thermo-protection buffer.

scholarly journals Whole genome sequencing of human enteroviruses from clinical samples by nanopore direct RNA sequencing

2020 ◽  
Author(s):  
C. Grädel ◽  
M.A. Terrazos Miani ◽  
C. Baumann ◽  
MT Barbani ◽  
S. Neuenschwander ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Rna Sequencing ◽  
Genome Sequencing ◽  
Illumina Miseq ◽  
High Viral Load ◽  
Clinical Samples ◽  
Whole Genome ◽  
Consensus Sequences ◽  
Positive Stool ◽  
Stool Samples

AbstractEnteroviruses are small RNA viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. In routine diagnostic laboratories, those viruses are identified by PCR based methods, often combined with partial sequencing for genotyping. In this proof-of-principle study, we assessed direct RNA sequencing (DRS) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. Results of the approach were complemented with those obtained by sequencing the corresponding viral cDNA via Illumina MiSeq sequencing. DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% to 99.6 % of the best reference genomes. The identification of the enterovirus sequences in the sample was confirmed by the short-read cDNA sequencing. Sequence identity between DRS and Illumina MiSeq enterovirus consensus sequences ranged between 94-97%. Here we show that nanopore DRS can be used to correctly identify the genotypes of enteroviruses from patient stool samples with high viral load.

Generating 1200bp tiled Amplicons for SARS-CoV2 Whole Genome Sequencing v1

2021 ◽  
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

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