scholarly journals A universal, single primer amplification protocol (R-SPA) to perform whole genome sequencing of segmented dsRNA reoviruses

2021 ◽  
Author(s):  
Klaudia Chrzastek ◽  
Holly S Sellers ◽  
Darrell Kapczynski
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Large Scale ◽  
Practical Method ◽  
Whole Genome ◽  
Amplification Method ◽  
Amplification Protocol ◽  
Wide Range ◽  
Genomic Characterization ◽  
Single Primer

Background. The Reoviridae family represents the largest family of double-stranded RNA (dsRNA) viruses, and the members have been isolated from a wide range of mammals, birds, reptiles, fishes, insects, plants. Orthoreoviruses, one of the 15 recognized genera in the Reoviridae family, can infect humans and nearly all mammals, and birds. Genomic characterization of reoviruses has not been adopted on a large-scale due to the complexity of obtaining sequences for all 10 segments. Results. In this study, we developed a time-efficient, and practical method to enrich reovirus sequencing reads from isolates that allowed for full genome recovery using single-primer amplification method coupled with next generation sequencing. We refer to this protocol as reovirus-Single Primer Amplification (R-SPA). Our results demonstrated that most of the genes were covered with at least 500 reads per base space. Furthermore, R-SPA covered both 5' and 3' end of each reovirus genes. Conclusion. A universal and fast amplification protocol that yields double-stranded cDNA in sufficient abundance and facilitates and expedites the whole genome sequencing of reoviruses was presented in this study.

scholarly journals Whole-genome sequencing of SARS-CoV-2 reveals the detection of G614 variant in Pakistan

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0248371
Author(s):  
Massab Umair ◽  
Aamer Ikram ◽  
Muhammad Salman ◽  
Adnan Khurshid ◽  
Masroor Alam ◽  
...  
Keyword(s):  
New England ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Large Scale ◽  
Genomic Diversity ◽  
Whole Genome ◽  
Whole Genome Analysis ◽  
Rapid Spread ◽  
Genomic Characterization ◽  
Amino Acid Mutations

Since its emergence in China, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide including Pakistan. During the pandemic, whole genome sequencing has played an important role in understanding the evolution and genomic diversity of SARS-CoV-2. Although an unprecedented number of SARS-CoV-2 full genomes have been submitted in GISAID and NCBI, data from Pakistan is scarce. We report the sequencing, genomic characterization, and phylogenetic analysis of five SARS-CoV-2 strains isolated from patients in Pakistan. The oropharyngeal swabs of patients that were confirmed positive for SARS-CoV-2 through real-time RT-PCR at National Institute of Health, Pakistan, were selected for whole-genome sequencing. Sequencing was performed using NEBNext Ultra II Directional RNA Library Prep kit for Illumina (NEW ENGLAND BioLabs Inc., MA, US) and Illumina iSeq 100 instrument (Illumina, San Diego, US). Based on whole-genome analysis, three Pakistani SARS-CoV-2 strains clustered into the 20A (GH) clade along with the strains from Oman, Slovakia, United States, and Pakistani strain EPI_ISL_513925. The two 19B (S)-clade strains were closely related to viruses from India and Oman. Overall, twenty-nine amino acid mutations were detected in the current study genome sequences, including fifteen missense and four novel mutations. Notably, we have found a D614G (aspartic acid to glycine) mutation in spike protein of the sequences from the GH clade. The G614 variant carrying the characteristic D614G mutation has been shown to be more infectious that lead to its rapid spread worldwide. This report highlights the detection of GH and S clade strains and G614 variant from Pakistan warranting large-scale whole-genome sequencing of strains prevalent in different regions to understand virus evolution and to explore their genetic diversity.

0306 Exploring the feasibility of using copy number variants as genetic markers through large-scale whole genome sequencing experiments

2016 ◽  
Vol 94 (suppl_5) ◽  
pp. 146-146
Author(s):  
D. M. Bickhart ◽  
L. Xu ◽  
J. L. Hutchison ◽  
J. B. Cole ◽  
D. J. Null ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genetic Markers ◽  
Genome Sequencing ◽  
Copy Number ◽  
Large Scale ◽  
Copy Number Variants ◽  
Whole Genome

scholarly journals Detection and characterization of copy number variants based on whole-genome sequencing by DNBSEQ platforms

2019 ◽  
Author(s):  
Junhua Rao ◽  
Lihua Peng ◽  
Fang Chen ◽  
Hui Jiang ◽  
Chunyu Geng ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Copy Number ◽  
Copy Number Variants ◽  
Copy Number Variant ◽  
Whole Genome ◽  
Genome Wide ◽  
Wide Range ◽  
Distribution Sensitivity ◽  
Cnv Detection

AbstractBackgroundNext-generation sequence (NGS) has rapidly developed in past years which makes whole-genome sequencing (WGS) becoming a more cost- and time-efficient choice in wide range of biological researches. We usually focus on some variant detection via WGS data, such as detection of single nucleotide polymorphism (SNP), insertion and deletion (Indel) and copy number variant (CNV), which playing an important role in many human diseases. However, the feasibility of CNV detection based on WGS by DNBSEQ™ platforms was unclear. We systematically analysed the genome-wide CNV detection power of DNBSEQ™ platforms and Illumina platforms on NA12878 with five commonly used tools, respectively.ResultsDNBSEQ™ platforms showed stable ability to detect slighter more CNVs on genome-wide (average 1.24-fold than Illumina platforms). Then, CNVs based on DNBSEQ™ platforms and Illumina platforms were evaluated with two public benchmarks of NA12878, respectively. DNBSEQ™ and Illumina platforms showed similar sensitivities and precisions on both two benchmarks. Further, the difference between tools for CNV detection was analyzed, and indicated the selection of tool for CNV detection could affected the CNV performance, such as count, distribution, sensitivity and precision.ConclusionThe major contribution of this paper is providing a comprehensive guide for CNV detection based on WGS by DNBSEQ™ platforms for the first time.

scholarly journals Improving tuberculosis surveillance by detecting international transmission using publicly available whole-genome sequencing data

2019 ◽  
Author(s):  
Andrea Sanchini ◽  
Christine Jandrasits ◽  
Julius Tembrockhaus ◽  
Thomas Andreas Kohl ◽  
Christian Utpatel ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Large Scale ◽  
Added Value ◽  
Whole Genome Sequencing Data ◽  
Whole Genome ◽  
International Transmission ◽  
The Public ◽  
Public Dataset ◽  
Public Repositories

AbstractIntroductionImproving the surveillance of tuberculosis (TB) is especially important for multidrug-resistant (MDR) and extensively drug-resistant (XDR)-TB. The large amount of publicly available whole-genome sequencing (WGS) data for TB gives us the chance to re-use data and to perform additional analysis at a large scale.AimWe assessed the usefulness of raw WGS data of global MDR/XDR-TB isolates available from public repositories to improve TB surveillance.MethodsWe extracted raw WGS data and the related metadata of Mycobacterium tuberculosis isolates available from the Sequence Read Archive. We compared this public dataset with WGS data and metadata of 131 MDR- and XDR-TB isolates from Germany in 2012-2013.ResultsWe aggregated a dataset that includes 1,081 MDR and 250 XDR isolates among which we identified 133 molecular clusters. In 16 clusters, the isolates were from at least two different countries. For example, cluster2 included 56 MDR/XDR isolates from Moldova, Georgia, and Germany. By comparing the WGS data from Germany and the public dataset, we found that 11 clusters contained at least one isolate from Germany and at least one isolate from another country. We could, therefore, connect TB cases despite missing epidemiological information.ConclusionWe demonstrated the added value of using WGS raw data from public repositories to contribute to TB surveillance. By comparing the German and the public dataset, we identified potential international transmission events. Thus, using this approach might support the interpretation of national surveillance results in an international context.

scholarly journals Population Structure of Mycobacterium bovis in Germany: a Long-Term Study Using Whole-Genome Sequencing Combined with Conventional Molecular Typing Methods

2020 ◽  
Vol 58 (11) ◽  
Author(s):  
Thomas A. Kohl ◽  
Katharina Kranzer ◽  
Sönke Andres ◽  
Thierry Wirth ◽  
Stefan Niemann ◽  
...  
Keyword(s):  
Population Structure ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Mycobacterium Bovis ◽  
Variable Number Tandem Repeat ◽  
Epidemiological Surveillance ◽  
Whole Genome ◽  
Content Type ◽  
Long Term Study ◽  
Wide Range

ABSTRACT Mycobacterium bovis is the primary cause of bovine tuberculosis (bTB) and infects a wide range of domestic animal and wildlife species and humans. In Germany, bTB still emerges sporadically in cattle herds, free-ranging wildlife, diverse captive animal species, and humans. In order to understand the underlying population structure and estimate the population size fluctuation through time, we analyzed 131 M. bovis strains from animals (n = 38) and humans (n = 93) in Germany from 1999 to 2017 by whole-genome sequencing (WGS), mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing, and spoligotyping. Based on WGS data analysis, 122 out of the 131 M. bovis strains were classified into 13 major clades, of which 6 contained strains from both human and animal cases and 7 only strains from human cases. Bayesian analyses suggest that the M. bovis population went through two sharp anticlimaxes, one in the middle of the 18th century and another one in the 1950s. WGS-based cluster analysis grouped 46 strains into 13 clusters ranging in size from 2 to 11 members and involving strains from distinct host types, e.g., only cattle and also mixed hosts. Animal strains of four clusters were obtained over a 9-year span, pointing toward autochthonous persistent bTB infection cycles. As expected, WGS had a higher discriminatory power than spoligotyping and MIRU-VNTR typing. In conclusion, our data confirm that WGS and suitable bioinformatics constitute the method of choice to implement prospective molecular epidemiological surveillance of M. bovis. The population of M. bovis in Germany is diverse, with subtle, but existing, interactions between different host groups.

scholarly journals Recombinational Switching of the Clostridium difficile S-Layer and a Novel Glycosylation Gene Cluster Revealed by Large-Scale Whole-Genome Sequencing

2012 ◽  
Vol 207 (4) ◽  
pp. 675-686 ◽  
Author(s):  
Kate E. Dingle ◽  
Xavier Didelot ◽  
M. Azim Ansari ◽  
David W. Eyre ◽  
Alison Vaughan ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Whole Genome Sequencing ◽  
Gene Cluster ◽  
Genome Sequencing ◽  
Large Scale ◽  
Whole Genome

scholarly journals Whole-Genome Sequencing for Routine Pathogen Surveillance in Public Health: a Population Snapshot of InvasiveStaphylococcus aureusin Europe

mBio ◽  
2016 ◽  
Vol 7 (3) ◽  
Author(s):  
David M. Aanensen ◽  
Edward J. Feil ◽  
Matthew T. G. Holden ◽  
Janina Dordel ◽  
Corin A. Yeats ◽  
...  
Keyword(s):  
Public Health ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Large Scale ◽  
Bacterial Pathogens ◽  
Epidemiological Surveillance ◽  
Data Sets ◽  
Whole Genome ◽  
Bioinformatic Tools ◽  
Road Map

ABSTRACTThe implementation of routine whole-genome sequencing (WGS) promises to transform our ability to monitor the emergence and spread of bacterial pathogens. Here we combined WGS data from 308 invasiveStaphylococcus aureusisolates corresponding to a pan-European population snapshot, with epidemiological and resistance data. Geospatial visualization of the data is made possible by a generic software tool designed for public health purposes that is available at the project URL (http://www.microreact.org/project/EkUvg9uY?tt=rc). Our analysis demonstrates that high-risk clones can be identified on the basis of population level properties such as clonal relatedness, abundance, and spatial structuring and by inferring virulence and resistance properties on the basis of gene content. We also show thatin silicopredictions of antibiotic resistance profiles are at least as reliable as phenotypic testing. We argue that this work provides a comprehensive road map illustrating the three vital components for future molecular epidemiological surveillance: (i) large-scale structured surveys, (ii) WGS, and (iii) community-oriented database infrastructure and analysis tools.IMPORTANCEThe spread of antibiotic-resistant bacteria is a public health emergency of global concern, threatening medical intervention at every level of health care delivery. Several recent studies have demonstrated the promise of routine whole-genome sequencing (WGS) of bacterial pathogens for epidemiological surveillance, outbreak detection, and infection control. However, as this technology becomes more widely adopted, the key challenges of generating representative national and international data sets and the development of bioinformatic tools to manage and interpret the data become increasingly pertinent. This study provides a road map for the integration of WGS data into routine pathogen surveillance. We emphasize the importance of large-scale routine surveys to provide the population context for more targeted or localized investigation and the development of open-access bioinformatic tools to provide the means to combine and compare independently generated data with publicly available data sets.

Sign in / Sign up

Export Citation Format

Share Document