A new approach to determining whole viral genomic sequences including termini using a single deep sequencing run

Alfson, Kendra J.; Beadles, Michael W.; Griffiths, Anthony John

doi:10.1016/j.jviromet.2014.07.023

Cited by 17 publications

(17 citation statements)

References 15 publications

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“…In this study, specimens with Ն4.5 log 10 copies/ml yielded approximately 5 million reads with 1.1% to 7.1% HIV-1 and full coverage at Ͼ600ϫ sequence depth (41). A noted advantage of the SMART technology is the ability to consistently sequence the 5= and 3= ends of viruses, challenging regions often missed by random-primed methods due to secondary structure (42)(43)(44). Therefore, the HIV-SMART ap-proach is an attractive, simple alternative that generally performs better than current unbiased priming methods.…”

Section: Discussionmentioning

confidence: 80%

A Pan-HIV Strategy for Complete Genome Sequencing

et al. 2016

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bMolecular surveillance is essential to monitor HIV diversity and track emerging strains. We have developed a universal library preparation method (HIV-SMART [i.e., switching mechanism at 5= end of RNA transcript]) for next-generation sequencing that harnesses the specificity of HIV-directed priming to enable full genome characterization of all HIV-1 groups (M, N, O, and P) and HIV-2. Broad application of the HIV-SMART approach was demonstrated using a panel of diverse cell-cultured virus isolates. HIV-1 non-subtype B-infected clinical specimens from Cameroon were then used to optimize the protocol to sequence directly from plasma. When multiplexing 8 or more libraries per MiSeq run, full genome coverage at a median ϳ2,000؋ depth was routinely obtained for either sample type. The method reproducibly generated the same consensus sequence, consistently identified viral sequence heterogeneity present in specimens, and at viral loads of <4.5 log copies/ml yielded sufficient coverage to permit strain classification. HIV-SMART provides an unparalleled opportunity to identify diverse HIV strains in patient specimens and to determine phylogenetic classification based on the entire viral genome. Easily adapted to sequence any RNA virus, this technology illustrates the utility of next-generation sequencing (NGS) for viral characterization and surveillance.

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Section: Discussionmentioning

confidence: 80%

A Pan-HIV Strategy for Complete Genome Sequencing

et al. 2016

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“…Isolation of viral RNA, sample preparation for deep sequencing, and deep sequencing were performed as previously described (29). Briefly, an aliquot of viral supernatant or serum was diluted in TRIzol LS reagent (Life Technologies).…”

Section: Ethicsmentioning

confidence: 99%

“…Samples were next transferred to a biosafety level 2 (BSL2) laboratory, where RNA was harvested following the manufacturer's instructions. After isolation, rRNA and mRNA were removed as described previously (29). Finally, the supernatant containing mRNA-depleted RNA was removed and prepared for deep sequencing using Illumina's TruSeq RNA sample preparation kit following the manufacturer's protocol.…”

Section: Ethicsmentioning

confidence: 99%

Determination and Therapeutic Exploitation of Ebola Virus Spontaneous Mutation Frequency

Alfson

Worwa

Carrión

et al. 2016

J Virol

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Ebola virus (EBOV) is an RNA virus that can cause hemorrhagic fever with high fatality rates, and there are no approved vaccines or therapies. Typically, RNA viruses have high spontaneous mutation rates, which permit rapid adaptation to selection pressures and have other important biological consequences. However, it is unknown if filoviruses exhibit high mutation frequencies. Ultradeep sequencing and a recombinant EBOV that carries the gene encoding green fluorescent protein were used to determine the spontaneous mutation frequency of EBOV. The effects of the guanosine analogue ribavirin during EBOV infections were also assessed. Ultradeep sequencing revealed that the mutation frequency for EBOV was high and similar to those of other RNA viruses. Interestingly, significant genetic diversity was not observed in viable viruses, implying that changes were not well tolerated. We hypothesized that this could be exploited therapeutically. In vitro, the presence of ribavirin increased the error rate, and the 50% inhibitory concentration (IC 50 ) was 27 M. In a mouse model of ribavirin therapy given pre-EBOV exposure, ribavirin treatment corresponded with a significant delay in time to death and up to 75% survival. In mouse and monkey models of therapy given post-EBOV exposure, ribavirin treatment also delayed the time to death and increased survival. These results demonstrate that EBOV has a spontaneous mutation frequency similar to those of other RNA viruses. These data also suggest a potential for therapeutic use of ribavirin for human EBOV infections. IMPORTANCEEbola virus (EBOV) causes a severe hemorrhagic disease with high case fatality rates; there are no approved vaccines or therapies. We determined the spontaneous mutation frequency of EBOV, which is relevant to understanding the potential for the virus to adapt. The frequency was similar to those of other RNA viruses. Significant genetic diversity was not observed in viable viruses, implying that changes were not well tolerated. We hypothesized that this could be exploited therapeutically. Ribavirin is a viral mutagen approved for treatment of several virus infections; it is also cheap and readily available. In cell culture, we showed that ribavirin was effective at reducing production of infectious EBOV. In mouse and monkey models of therapy given post-EBOV exposure, ribavirin treatment delayed the time to death and increased survival. These data provide a better understanding of EBOV spontaneous mutation and suggest that ribavirin may have great value in the context of human disease.

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“…Nearly all of the short missing regions occurred at either the termini (a common issue in viral NGS sequencing) [46] or at one specific intergenic location in the genome between genes N and P which displayed extremely low coverage in all analyzed samples (possibly as a result of high GC content – 76%). For the purpose of submitting full-length NDV sequences to GenBank, we sequenced the termini using a previously described protocol [47] and primers designed for the current study (see Additional file 4: Table S3).…”

Section: Resultsmentioning

confidence: 99%

A robust and cost-effective approach to sequence and analyze complete genomes of small RNA viruses

Dimitrov

Sharma

Volkening

et al. 2017

Virol J

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BackgroundNext-generation sequencing (NGS) allows ultra-deep sequencing of nucleic acids. The use of sequence-independent amplification of viral nucleic acids without utilization of target-specific primers provides advantages over traditional sequencing methods and allows detection of unsuspected variants and co-infecting agents. However, NGS is not widely used for small RNA viruses because of incorrectly perceived cost estimates and inefficient utilization of freely available bioinformatics tools.MethodsIn this study, we have utilized NGS-based random sequencing of total RNA combined with barcode multiplexing of libraries to quickly, effectively and simultaneously characterize the genomic sequences of multiple avian paramyxoviruses. Thirty libraries were prepared from diagnostic samples amplified in allantoic fluids and their total RNAs were sequenced in a single flow cell on an Illumina MiSeq instrument. After digital normalization, data were assembled using the MIRA assembler within a customized workflow on the Galaxy platform.ResultsTwenty-eight avian paramyxovirus 1 (APMV-1), one APMV-13, four avian influenza and two infectious bronchitis virus complete or nearly complete genome sequences were obtained from the single run. The 29 avian paramyxovirus genomes displayed 99.6% mean coverage based on bases with Phred quality scores of 30 or more. The lower and upper quartiles of sample median depth per position for those 29 samples were 2984 and 6894, respectively, indicating coverage across samples sufficient for deep variant analysis. Sample processing and library preparation took approximately 25–30 h, the sequencing run took 39 h, and processing through the Galaxy workflow took approximately 2–3 h. The cost of all steps, excluding labor, was estimated to be 106 USD per sample.ConclusionsThis work describes an efficient multiplexing NGS approach, a detailed analysis workflow, and customized tools for the characterization of the genomes of RNA viruses. The combination of multiplexing NGS technology with the Galaxy workflow platform resulted in a fast, user-friendly, and cost-efficient protocol for the simultaneous characterization of multiple full-length viral genomes. Twenty-nine full-length or near-full-length APMV genomes with a high median depth were successfully sequenced out of 30 samples. The applied de novo assembly approach also allowed identification of mixed viral populations in some of the samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-017-0741-5) contains supplementary material, which is available to authorized users.

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A new approach to determining whole viral genomic sequences including termini using a single deep sequencing run

Cited by 17 publications

References 15 publications

A Pan-HIV Strategy for Complete Genome Sequencing

A Pan-HIV Strategy for Complete Genome Sequencing

Determination and Therapeutic Exploitation of Ebola Virus Spontaneous Mutation Frequency

A robust and cost-effective approach to sequence and analyze complete genomes of small RNA viruses

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