scholarly journals Development and Validation of a High-Throughput Next-Generation Sequencing Workflow for SARS-CoV-2 Whole Genome Sequencing: Results from Over 65,000 Clinical Cases

2021 ◽  
Author(s):  
Sun Hee Rosenthal ◽  
Anna Gerasimova ◽  
Rolando Ruiz-Vega ◽  
Kayla Livingston ◽  
Ron M. Kagan ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
High Throughput ◽  
The United States ◽  
Primer Binding Site ◽  
Whole Genome ◽  
Next Generation ◽  
Pcr Method ◽  
Generation Sequencing

Abstract Monitoring new mutations in SARS-CoV-2 provides crucial information for identifying diagnostic and therapeutic targets and important insights to achieve a more effective COVID-19 control strategy. Next generation sequencing (NGS) technologies have been widely used for whole genome sequencing of SARS-CoV-2. While various NGS methods have been reported, one chief limitation has been the complexity of the workflow, limiting the scalability. Here, we overcome this limitation by designing a workflow optimized for high-throughput studies. The workflow utilizes modified ARTIC network v3 primers for SARS-CoV-2 whole genome amplification. NGS libraries were prepared by a 2-step PCR method, similar to a previously reported tailed PCR method, with further optimizations to improve amplicon balance, to minimize amplicon dropout for viral genomes harboring primer-binding site mutation(s), and to integrate robotic liquid handlers. Validation studies demonstrated that the optimized workflow can process up to 2,688 samples in a single sequencing run without compromising sensitivity and accuracy and with fewer amplicon dropout events compared to the standard ARTIC protocol. We additionally report results for over 65,000 SARS-CoV-2 whole genome sequences from clinical specimens collected in the United States between January and September of 2021, as part of an ongoing national genomics surveillance effort.

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

2019 ◽  
Vol 24 (2) ◽  
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.

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 Whole genome sequencing of Indonesian dengue virus isolates using next-generation sequencing

2018 ◽  
Vol 23 (2) ◽  
pp. 74
Author(s):  
Benediktus Yohan ◽  
Rama Dhenni ◽  
Rahma F Hayati ◽  
Frilasita Aisyah Yudhaputri ◽  
Dionisius Denis ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Vaccine Development ◽  
Antiviral Drug ◽  
Clinical Aspects ◽  
High Morbidity ◽  
Whole Genome ◽  
Next Generation ◽  
Generation Sequencing

Indonesia is a tropical country and hyperendemic for dengue. The disease prevalently affected Indonesian and it caused high morbidity and substantial economic burden. This vector-borne viral disease is caused by infection of dengue viruses (DENVs), which are the member of Flaviviridae family. While most of dengue studies in Indonesia focused on the epidemiology, the clinical aspects, the vectors, and to certain extent the virology, there were still gaps in the DENVs genomic aspects. Considering their high mutation rate, the DENVs were known for their high genetic diversity and it might affect the characteristics of the viruses. Comprehensive DENV genomic data were thus important for many aspects of disease management, including virus surveillance, pathogenesis, diagnostics, antiviral drug design, and vaccine development. We established in this study a method for DENV whole genome sequencing using the advanced Next-Generation Sequencing (NGS) and Nextera XT DNA library preparation kit, coupled with simplified bioinformatic analysis methods. The Indonesian DENVs from four serotypes were isolated from patients’ sera, while library was prepared from enriched templates and sequenced using Illumina NGS. Our study highlighted the potential of a robust NGS method in producing whole genome sequence of DENVs, which would be important for future dengue studies.

scholarly journals Fighting an old disease with next-generation sequencing

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Anzaan Dippenaar ◽  
Robin M Warren
Keyword(s):  
Next Generation Sequencing ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Next Generation ◽  
High Incidence ◽  
Generation Sequencing

Whole genome sequencing has revealed that most cases of tuberculosis in a high-incidence setting in Malawi were caused by just one lineage of the bacterium responsible for the disease.

scholarly journals A novel algorithm comprehensively characterizes human RH genes using whole-genome sequencing data

2020 ◽  
Vol 4 (18) ◽  
pp. 4347-4357
Author(s):  
Ti-Cheng Chang ◽  
Kelly M. Haupfear ◽  
Jing Yu ◽  
Evadnie Rampersaud ◽  
Vivien A. Sheehan ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Next Generation Sequencing Data ◽  
Blood Group Antigens ◽  
Whole Genome ◽  
Next Generation ◽  
Sequencing Data ◽  
Generation Sequencing ◽  
Novel Algorithm

Abstract RHD and RHCE genes encode Rh blood group antigens and exhibit extensive single-nucleotide polymorphisms and chromosome structural changes in patients with sickle cell disease (SCD). RH variation can drive loss of antigen epitopes or expression of new epitopes, predisposing patients with SCD to Rh alloimmunization. Serologic antigen typing is limited to common Rh antigens, necessitating a genetic approach to detect variant antigen expression. We developed a novel algorithm termed RHtyper for RH genotyping from existing whole-genome sequencing (WGS) data. RHtyper determined RH genotypes in an average of 3.4 and 3.3 minutes per sample for RHD and RHCE, respectively. In a validation cohort consisting of 57 patients with SCD, RHtyper achieved 100% accuracy for RHD and 98.2% accuracy for RHCE, when compared with genotypes obtained by RH BeadChip and targeted molecular assays and after verification by Sanger sequencing and independent next-generation sequencing assays. RHtyper was next applied to WGS data from an additional 827 patients with SCD. In the total cohort of 884 patients, RHtyper identified 38 RHD and 28 RHCE distinct alleles, including a novel RHD DAU allele, RHD* 602G, 733C, 744T 1136T. RHtyper provides comprehensive and high-throughput RH genotyping from WGS data, facilitating deconvolution of the extensive RH genetic variation among patients with SCD. We have implemented RHtyper as a cloud-based public access application in DNAnexus (https://platform.dnanexus.com/app/RHtyper), enabling clinicians and researchers to perform RH genotyping with next-generation sequencing data.

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