Sequencing Using a Two-Step Strategy Reveals High Genetic Diversity in the S Gene of SARS-CoV-2 after a High-Transmission Period in Tunis, Tunisia

Recent efforts have reported numerous variants that influence SARS-CoV-2 viral characteristics including pathogenicity, transmission rate and ability of detection by molecular tests. Whole genome sequencing based on NGS technologies is the method of choice to identify all viral variants; however, the resources needed to use these techniques for a representative number of specimens remain limited in many low and middle income countries. To decrease sequencing cost, we developed a couple of primers allowing to generate partial sequences in the viral S gene allowing rapid detection of numerous variants of concern (VOCs) and variants of interest (VOIs); whole genome sequencing is then performed on a selection of viruses based on partial sequencing results. Two hundred and one nasopharyngeal specimens collected during the decreasing phase of a high transmission COVID-19 wave in Tunisia were analyzed. The results reveal high genetic variability within the sequenced fragment and allowed the detection of first introduction in the country of already known VOCs and VOIs as well as others variants that have interesting genomic mutations and need to be kept under surveillance.

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Whole genome sequencing suggests transmission of Corynebacterium diphtheriae-caused cutaneous diphtheria in two siblings, Germany, 2018

Eurosurveillance ◽

10.2807/1560-7917.es.2019.24.2.1800683 ◽

2019 ◽

Vol 24 (2) ◽

Cited By ~ 3

Author(s):

Anja Berger ◽

Alexandra Dangel ◽

Tilmann Schober ◽

Birgit Schmidbauer ◽

Regina Konrad ◽

...

Keyword(s):

Next Generation Sequencing ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Corynebacterium Diphtheriae ◽

Whole Genome ◽

Next Generation ◽

Insect Bites ◽

Next Generation Sequencing Ngs ◽

Ngs Data ◽

Generation Sequencing

In September 2018, a child who had returned from Somalia to Germany presented with cutaneous diphtheria by toxigenic Corynebacterium diphtheriae biovar mitis. The child’s sibling had superinfected insect bites harbouring also toxigenic C. diphtheriae. Next generation sequencing (NGS) revealed the same strain in both patients suggesting very recent human-to-human transmission. Epidemiological and NGS data suggest that the two cutaneous diphtheria cases constitute the first outbreak by toxigenic C. diphtheriae in Germany since the 1980s.

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Rapid in-country sequencing of whole virus genomes to inform rabies elimination programmes

Wellcome Open Research ◽

10.12688/wellcomeopenres.15518.2 ◽

2020 ◽

Vol 5 ◽

pp. 3 ◽

Cited By ~ 3

Author(s):

Kirstyn Brunker ◽

Gurdeep Jaswant ◽

S.M. Thumbi ◽

Kennedy Lushasi ◽

Ahmed Lugelo ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genomic Data ◽

Genomic Research ◽

Whole Genome ◽

Middle Income ◽

Endemic Disease ◽

Middle Income Countries ◽

Virus Genomes ◽

Low And Middle Income

Genomic surveillance is an important aspect of contemporary disease management but has yet to be used routinely to monitor endemic disease transmission and control in low- and middle-income countries. Rabies is an almost invariably fatal viral disease that causes a large public health and economic burden in Asia and Africa, despite being entirely vaccine preventable. With policy efforts now directed towards achieving a global goal of zero dog-mediated human rabies deaths by 2030, establishing effective surveillance tools is critical. Genomic data can provide important and unique insights into rabies spread and persistence that can direct control efforts. However, capacity for genomic research in low- and middle-income countries is held back by limited laboratory infrastructure, cost, supply chains and other logistical challenges. Here we present and validate an end-to-end workflow to facilitate affordable whole genome sequencing for rabies surveillance utilising nanopore technology. We used this workflow in Kenya, Tanzania and the Philippines to generate rabies virus genomes in two to three days, reducing costs to approximately £60 per genome. This is over half the cost of metagenomic sequencing previously conducted for Tanzanian samples, which involved exporting samples to the UK and a three- to six-month lag time. Ongoing optimization of workflows are likely to reduce these costs further. We also present tools to support routine whole genome sequencing and interpretation for genomic surveillance. Moreover, combined with training workshops to empower scientists in-country, we show that local sequencing capacity can be readily established and sustainable, negating the common misperception that cutting-edge genomic research can only be conducted in high resource laboratories. More generally, we argue that the capacity to harness genomic data is a game-changer for endemic disease surveillance and should precipitate a new wave of researchers from low- and middle-income countries.

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Implementation of whole genome sequencing for tuberculosis diagnostics in a low-middle income, high MDR-TB burden country

Scientific Reports ◽

10.1038/s41598-021-94297-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Monica Vogel ◽

Christian Utpatel ◽

Caroline Corbett ◽

Thomas A. Kohl ◽

Altyn Iskakova ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Successful Implementation ◽

Whole Genome ◽

Kyrgyz Republic ◽

Middle Income ◽

External Assessment ◽

Middle Income Countries ◽

Mdr Tb ◽

Miseq Platform

AbstractWhole genome sequencing (WGS) is revolutionary for diagnostics of TB and its mutations associated with drug-resistances, but its uptake in low- and middle-income countries is hindered by concerns of implementation feasibility. Here, we provide a proof of concept for its successful implementation in such a setting. WGS was implemented in the Kyrgyz Republic. We estimated needs of up to 55 TB-WGS per week and chose the MiSeq platform (Illumina, USA) because of its capacity of up to 60 TB-WGS per week. The project’s timeline was completed in 93-weeks. Costs of large equipment and accompanying costs were 222,065 USD and 8462 USD, respectively. The first 174 WGS costed 277 USD per sequence, but this was skewed by training inefficiencies. Based on real prices and presuming optimal utilization of WGS capacities, WGS costs could drop to 167 and 141 USD per WGS using MiSeq Reagent Kits v2 (500-cycles) and v3 (600-cycles), respectively. Five trainings were required to prepare the staff for autonomous WGS which cost 48,250 USD. External assessment confirmed excellent performance of WGS by the Kyrgyz laboratory in an interlaboratory comparison of 30 M. tuberculosis genomes showing complete agreeance of results.

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Rapid in-country sequencing of whole virus genomes to inform rabies elimination programmes

Wellcome Open Research ◽

10.12688/wellcomeopenres.15518.1 ◽

2020 ◽

Vol 5 ◽

pp. 3

Author(s):

Kirstyn Brunker ◽

Gurdeep Jaswant ◽

S.M. Thumbi ◽

Kennedy Lushasi ◽

Ahmed Lugelo ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genomic Data ◽

Genomic Research ◽

Whole Genome ◽

Middle Income ◽

Endemic Disease ◽

Middle Income Countries ◽

Virus Genomes ◽

Low And Middle Income

Genomic surveillance is an important aspect of contemporary disease management but has yet to be used routinely to monitor endemic disease transmission and control in low- and middle-income countries. Rabies is an almost invariably fatal viral disease that causes a large public health and economic burden in Asia and Africa, despite being entirely vaccine preventable. With policy efforts now directed towards achieving a global goal of zero dog-mediated human rabies deaths by 2030, establishing effective surveillance tools is critical. Genomic data can provide important and unique insights into rabies spread and persistence that can direct control efforts. However, capacity for genomic research in low- and middle-income countries is held back by limited laboratory infrastructure, cost, supply chains and other logistical challenges. Here we present and validate an end-to-end workflow to facilitate affordable whole genome sequencing for rabies surveillance utilising nanopore technology. We used this workflow in Kenya, Tanzania and the Philippines to generate rabies virus genomes in two to three days, reducing costs to approximately £60 per genome. This is over half the cost of metagenomic sequencing previously conducted for Tanzanian samples, which involved exporting samples to the UK and a three- to six-month lag time. Ongoing optimization of workflows are likely to reduce these costs further. We also present tools to support routine whole genome sequencing and interpretation for genomic surveillance. Moreover, combined with training workshops to empower scientists in-country, we show that local sequencing capacity can be readily established and sustainable, negating the common misperception that cutting-edge genomic research can only be conducted in high resource laboratories. More generally, we argue that the capacity to harness genomic data is a game-changer for endemic disease surveillance and should precipitate a new wave of researchers from low- and middle-income countries.

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A Preterm Infant with Multiple Anomalies Diagnosed with Atypical CHARGE Syndrome after a Novel CHD7 Variant Confirmed Using Whole-Genome Sequencing

Neonatology ◽

10.1159/000506165 ◽

2020 ◽

Vol 117 (3) ◽

pp. 374-379

Author(s):

Hyun Ho Kim ◽

Ah Reum Kim ◽

Nayoung K.D. Kim ◽

So Yoon Ahn ◽

Se In Sung ◽

...

Keyword(s):

Preterm Infant ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Broad Spectrum ◽

De Novo ◽

Charge Syndrome ◽

Whole Genome ◽

Life Threatening ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

CHARGE syndrome has a clinically broad spectrum of phenotypes, including partial or atypical type. CHD7 mutation is related to CHARGE syndrome that shows various phenotypes according to the CHD7 variant. Developments in genetic analysis techniques, such as next-generation sequencing (NGS), are helping both diagnosis and treatment of diseases. We report the case of a preterm infant diagnosed with atypical CHARGE who has a novel and de novo CHD7 variant that was identified using whole-genome sequencing (WGS). Neonatologists tend to be reluctant to diagnose infants with multiple malformations because they have to focus on treating life-threatening complications; however, NGS is considered helpful for the early diagnosis of broad-spectrum anomalies during the neonatal period.

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Application of next generation sequencing (NGS) system for whole-genome sequencing of porcine reproductive and respiratory syndrome virus (PRRSV)

Korean Journal of Veterinary Service ◽

10.7853/kjvs.2016.39.1.41 ◽

2016 ◽

Vol 39 (1) ◽

pp. 41-49

Author(s):

Sung-Hyun Moon ◽

Amina Khatun ◽

Won-Il Kim ◽

Md Mukter Hossain ◽

Yeonsu Oh ◽

...

Keyword(s):

Next Generation Sequencing ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Respiratory Syndrome Virus ◽

Whole Genome ◽

Next Generation ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

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

Scientific Reports ◽

10.1038/s41598-019-53043-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 5

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.

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Rapid whole genome sequencing of Miyazaki-Bali/2007 Pteropine orthoreovirus by modified rolling circular amplification with adaptor ligation – next generation sequencing

Scientific Reports ◽

10.1038/srep16517 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 7

Author(s):

Harpal Singh ◽

Tomoki Yoshikawa ◽

Takeshi Kobayashi ◽

Shuetsu Fukushi ◽

Hideki Tani ◽

...

Keyword(s):

Next Generation Sequencing ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome ◽

Next Generation ◽

Generation Sequencing

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Whole Genome Sequencing of Nontuberculous Mycobacterium (NTM) Isolates from Sputum and Environmental Specimens in Co-Habiting Patients with NTM Pulmonary Disease

10.21203/rs.2.17722/v1 ◽

2019 ◽

Author(s):

Jung-Ki Yoon ◽

Taek Soo Kim ◽

Jong-Il Kim ◽

Jae-Joon Yim

Keyword(s):

Whole Genome Sequencing ◽

Pulmonary Disease ◽

Genome Sequencing ◽

Genetic Distances ◽

Mycobacterium Abscessus ◽

Nontuberculous Mycobacterium ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Next Generation Sequencing Ngs

Abstract Background : Nontuberculous mycobacterium (NTM) species are ubiquitous microorganisms. NTM pulmonary disease (NTM-PD) is caused not by human-to-human transmission but by independent environmental acquisition. However, recent studies using next-generation sequencing (NGS) have reported trans-continental spread of Mycobacterium abscessus among patients with cystic fibrosis. Results : We investigated NTM genomes through NGS to examine transmission patterns in three pairs of co-habiting NTM-PD patients who were suspected of patient-to-patient transmission. Three pairs of patients with NTM-PD co-habiting for at least 15 years were enrolled: a mother and a daughter with M. avium PD, a couple with M. intracellulare PD, and a second couple, one of whom was infected with M. intracellulare PD and the other of whom was infected with M. abscessus subsp. massiliense PD. Whole genome sequencing was performed using NTM colonies isolated from patients and environmental specimens. Genetic distances were estimated based on single nucleotide polymorphisms (SNPs) in the NTM genomes. Comparing SNPs in the consensus regions, the minimum pairwise SNP distances of NTM isolates derived from the two pairs of patients infected with the same NTM species were over 10,000. In phylogenetic analysis, the NTM isolates from patients with M. avium PD clustered with isolates from different environmental sources. Conclusions : In conclusion, considering the genetic distances between NTM strains, the likelihood of patient-to-patient transmission in pairs of co-habiting NTM-PD patients without overt immune deficiency is minimal.

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