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Review
. 2021 Feb 22:12:621719.
doi: 10.3389/fmicb.2021.621719. eCollection 2021.

High Throughput Sequencing for the Detection and Characterization of RNA Viruses

Amy H Fitzpatrick  1   2   3 , Agnieszka Rupnik  2 , Helen O'Shea  3 , Fiona Crispie  1 , Sinéad Keaveney  2 , Paul Cotter  1
Affiliations
Review

High Throughput Sequencing for the Detection and Characterization of RNA Viruses

Amy H Fitzpatrick et al. Front Microbiol. .

Abstract

This review aims to assess and recommend approaches for targeted and agnostic High Throughput Sequencing of RNA viruses in a variety of sample matrices. HTS also referred to as deep sequencing, next generation sequencing and third generation sequencing; has much to offer to the field of environmental virology as its increased sequencing depth circumvents issues with cloning environmental isolates for Sanger sequencing. That said however, it is important to consider the challenges and biases that method choice can impart to sequencing results. Here, methodology choices from RNA extraction, reverse transcription to library preparation are compared based on their impact on the detection or characterization of RNA viruses.

Keywords: RNA depletion; RNA viruses; amplicon sequencing; capture based probe hybridization; environmental virology; high throughput sequencing; viral enrichment.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Targeted HTS approaches.
Figure 2
Figure 2
Enrichment methods for agnostic sequencing.
Figure 3
Figure 3
rRNA depletion methods for agnostic sequencing.
Figure 4
Figure 4
Decision tree guiding method choices for HTS of RNA viruses.
See this image and copyright information in PMC

References

    1. Aarem J., Brunborg G., Aas K. K., Harbak K., Taipale M. M., Magnus P., et al. . (2016). Comparison of blood RNA isolation methods from samples stabilized in Tempus tubes and stored at a large human biobank. BMC Res. 9:430. 10.1186/s13104-016-2224-y - DOI - PMC - PubMed
    1. Acevedo A., Brodsky L., Andino R. (2014). Mutational and fitness landscapes of an RNA virus revealed through population sequencing. Nature 505, 686–690. 10.1038/nature12861 - DOI - PMC - PubMed
    1. Adams N. M., Bordelon H., Wang K.-K. A., Albert L. E., Wright D. W., Haselton F. R. (2015). Comparison of three magnetic bead surface functionalities for RNA extraction and detection. ACS Appl. Mater. Interfaces 7, 6062–6069. 10.1021/am506374t - DOI - PubMed
    1. Adiconis X., Borges-Rivera D., Satija R., DeLuca D. S., Busby M. A., Berlin A. M., et al. . (2013). Comparative analysis of RNA sequencing methods for degraded or low-input samples. Nat. Methods 10, 623–629. 10.1038/nmeth.2483 - DOI - PMC - PubMed
    1. Ahmed R., Hossain M. S., Haque M. S., Alam M. M., Islam M. S. (2019). Modified protocol for RNA isolation from different parts of field-grown jute plant suitable for NGS data generation and quantitative real-time RT-PCR. Afr. J. Biotechnol. 18, 647–658. 10.5897/AJB2019.16819 - DOI

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