Whole genome sequencing of respiratory syncytial (RSV) virus from clinical samples with low viral load v1 (protocols.io.qsedwbe)

The genomic surveillance of porcine reproductive and respiratory syndrome virus (PRRSV) is based on sequencing of the ORF5 gene of the virus, which covers only 4% of the entire viral genome. It is expected that PRRSV whole-genome sequencing (WGS) will improve PRRSV genomic data and allow better understanding of clinical discrepancies observed in the field when using ORF5 sequencing. Our main objective was to implement an efficient method for WGS of PRRSV from clinical samples. The viral genome was purified using a poly(A)-tail viral genome purification method and sequenced using Illumina technology. We tested 149 PRRSV-positive samples: 80 sera, 33 lungs, 33 pools of tissues, 2 oral fluids, and 1 processing fluid (i.e., castration liquid). Overall, WGS of 67.1% of PRRSV-positive cases was successful. The viral load, in particular for tissues, had a major impact on the PRRSV WGS success rate. Serum was the most efficient type of sample to conduct PRRSV WGS poly(A)-tail assays, with a success rate of 76.3%, and this result can be explained by improved sequencing reads dispersion matching throughout the entire viral genome. WGS was unsuccessful for all pools of tissue and lung samples with Cq values > 26.5, whereas it could still be successful with sera at Cq ≤ 34.1. Evaluation of results of highly qualified personnel confirmed that laboratory skills could affect PRRSV WGS efficiency. Oral fluid samples seem very promising and merit further investigation because, with only 2 samples of low viral load (Cq = 28.8, 32.8), PRRSV WGS was successful.

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Cattle connection: molecular epidemiology of BVDV outbreaks via rapid nanopore whole-genome sequencing of clinical samples

BMC Veterinary Research ◽

10.1186/s12917-021-02945-3 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Jacqueline King ◽

Anne Pohlmann ◽

Kamila Dziadek ◽

Martin Beer ◽

Kerstin Wernike

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Economic Losses ◽

Clinical Samples ◽

Bovine Viral Diarrhea ◽

Sequence Information ◽

Whole Genome ◽

Diarrhea Virus ◽

Viral Diarrhea ◽

Viral Loads

Abstract Background As a global ruminant pathogen, bovine viral diarrhea virus (BVDV) is responsible for the disease Bovine Viral Diarrhea with a variety of clinical presentations and severe economic losses worldwide. Classified within the Pestivirus genus, the species Pestivirus A and B (syn. BVDV-1, BVDV-2) are genetically differentiated into 21 BVDV-1 and four BVDV-2 subtypes. Commonly, the 5’ untranslated region and the Npro protein are utilized for subtyping. However, the genetic variability of BVDV leads to limitations in former studies analyzing genome fragments in comparison to a full-genome evaluation. Results To enable rapid and accessible whole-genome sequencing of both BVDV-1 and BVDV-2 strains, nanopore sequencing of twelve representative BVDV samples was performed on amplicons derived through a tiling PCR procedure. Covering a multitude of subtypes (1b, 1d, 1f, 2a, 2c), sample matrices (plasma, EDTA blood and ear notch), viral loads (Cq-values 19–32) and species (cattle and sheep), ten of the twelve samples produced whole genomes, with two low titre samples presenting 96 % genome coverage. Conclusions Further phylogenetic analysis of the novel sequences emphasizes the necessity of whole-genome sequencing to identify novel strains and supplement lacking sequence information in public repositories. The proposed amplicon-based sequencing protocol allows rapid, inexpensive and accessible obtainment of complete BVDV genomes.

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Identification of low abundance microbiome in clinical samples using whole genome sequencing

Genome Biology ◽

10.1186/s13059-015-0821-z ◽

2015 ◽

Vol 16 (1) ◽

Cited By ~ 55

Author(s):

Chao Zhang ◽

Kyle Cleveland ◽

Felice Schnoll-Sussman ◽

Bridget McClure ◽

Michelle Bigg ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Clinical Samples ◽

Whole Genome

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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 for Detection and Characterization of Microorganisms Directly from Clinical Samples

Journal of Clinical Microbiology ◽

10.1128/jcm.01369-14 ◽

2014 ◽

Vol 52 (8) ◽

pp. 3136-3136 ◽

Cited By ~ 5

Author(s):

H. Hasman ◽

D. Saputra ◽

T. Sicheritz-Ponten ◽

O. Lund ◽

C. A. Svendsen ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Clinical Samples ◽

Whole Genome

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

Journal of Clinical Microbiology ◽

10.1128/jcm.03073-14 ◽

2015 ◽

Vol 53 (4) ◽

pp. 1137-1143 ◽

Cited By ~ 47

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.

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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

10.1101/355560 ◽

2018 ◽

Cited By ~ 2

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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Discordant bioinformatic predictions of antimicrobial resistance from whole-genome sequencing data of bacterial isolates: An inter-laboratory study

10.1101/793885 ◽

2019 ◽

Cited By ~ 1

Author(s):

Ronan M. Doyle ◽

Denise M. O’Sullivan ◽

Sean D. Aller ◽

Sebastian Bruchmann ◽

Taane Clark ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Laboratory Study ◽

Clinical Microbiology ◽

Sequence Data ◽

Clinical Samples ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Sequencing Data

AbstractBackgroundAntimicrobial resistance (AMR) poses a threat to public health. Clinical microbiology laboratories typically rely on culturing bacteria for antimicrobial susceptibility testing (AST). As the implementation costs and technical barriers fall, whole-genome sequencing (WGS) has emerged as a ‘one-stop’ test for epidemiological and predictive AST results. Few published comparisons exist for the myriad analytical pipelines used for predicting AMR. To address this, we performed an inter-laboratory study providing sets of participating researchers with identical short-read WGS data sequenced from clinical isolates, allowing us to assess the reproducibility of the bioinformatic prediction of AMR between participants and identify problem cases and factors that lead to discordant results.MethodsWe produced ten WGS datasets of varying quality from cultured carbapenem-resistant organisms obtained from clinical samples sequenced on either an Illumina NextSeq or HiSeq instrument. Nine participating teams (‘participants’) were provided these sequence data without any other contextual information. Each participant used their own pipeline to determine the species, the presence of resistance-associated genes, and to predict susceptibility or resistance to amikacin, gentamicin, ciprofloxacin and cefotaxime.ResultsIndividual participants predicted different numbers of AMR-associated genes and different gene variants from the same clinical samples. The quality of the sequence data, choice of bioinformatic pipeline and interpretation of the results all contributed to discordance between participants. Although much of the inaccurate gene variant annotation did not affect genotypic resistance predictions, we observed low specificity when compared to phenotypic AST results but this improved in samples with higher read depths. Had the results been used to predict AST and guide treatment a different antibiotic would have been recommended for each isolate by at least one participant.ConclusionsWe found that participants produced discordant predictions from identical WGS data. These challenges, at the final analytical stage of using WGS to predict AMR, suggest the need for refinements when using this technology in clinical settings. Comprehensive public resistance sequence databases and standardisation in the comparisons between genotype and resistance phenotypes will be fundamental before AST prediction using WGS can be successfully implemented in standard clinical microbiology laboratories.

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Efficient whole genome sequencing of influenza A viruses

10.1101/749234 ◽

2019 ◽

Author(s):

Marina Escalera-Zamudio ◽

Ana Georgina Cobián-Güemes ◽

Blanca Taboada ◽

Irma López-Martínez ◽

Joel Armando Vázquez-Pérez ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Influenza A Virus ◽

Genome Sequencing ◽

Influenza A ◽

Genetic Material ◽

Influenza Viruses ◽

Epidemiological Surveillance ◽

Clinical Samples ◽

Whole Genome ◽

Influenza A Viruses

ABSTRACTThe constant threat of emergence for novel pathogenic influenza A viruses with pandemic potential, makes full-genome characterization of circulating influenza viral strains a high priority, allowing detection of novel and re-assorting variants. Sequencing the full-length genome of influenza A virus traditionally required multiple amplification rounds, followed by the subsequent sequencing of individual PCR products. The introduction of high-throughput sequencing technologies has made whole genome sequencing easier and faster. We present a simple protocol to obtain whole genome sequences of hypothetically any influenza A virus, even with low quantities of starting genetic material. The complete genomes of influenza A viruses of different subtypes and from distinct sources (clinical samples of pdmH1N1, tissue culture-adapted H3N2 viruses, or avian influenza viruses from cloacal swabs) were amplified with a single multisegment reverse transcription-PCR reaction and sequenced using Illumina sequencing platform. Samples with low quantity of genetic material after initial PCR amplification were re-amplified by an additional PCR using random primers. Whole genome sequencing was successful for 66% of the samples, whilst the most relevant genome segments for epidemiological surveillance (corresponding to the hemagglutinin and neuraminidase) were sequenced with at least 93% coverage (and a minimum 10x) for 98% of the samples. Low coverage for some samples is likely due to an initial low viral RNA concentration in the original sample. The proposed methodology is especially suitable for sequencing a large number of samples, when genetic data is urgently required for strains characterization, and may also be useful for variant analysis.

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