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Library preparation for highly accurate population sequencing of RNA viruses

Nature Protocols volume 9, pages 1760–1769 (2014)Cite this article

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Abstract

Circular resequencing (CirSeq) is a novel technique for efficient and highly accurate next-generation sequencing (NGS) of RNA virus populations. The foundation of this approach is the circularization of fragmented viral RNAs, which are then redundantly encoded into tandem repeats by 'rolling-circle' reverse transcription. When sequenced, the redundant copies within each read are aligned to derive a consensus sequence of their initial RNA template. This process yields sequencing data with error rates far below the variant frequencies observed for RNA viruses, facilitating ultra-rare variant detection and accurate measurement of low-frequency variants. Although library preparation takes ∼5 d, the high-quality data generated by CirSeq simplifies downstream data analysis, making this approach substantially more tractable for experimentalists.

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Figure 1: Schematic of CirSeq.
Figure 2: Analysis of coverage from libraries produced with different-sized RNA fragments.
Figure 3: Analysis of variant frequency error.
Figure 4: Bioanalysis of size-selected fragmented RNA.

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Acknowledgements

We thank S. Taguwa and P. Ambrose for critical reading of the manuscript, and G. Schroth, M. Harrison, P. Wassam and T. Collins for technical advice. This work was financially supported by a National Science Foundation graduate research fellowship to A.A., and by National Institute of Allergy and Infectious Diseases (NIAID) grants AI091575, AI36178 and AI40085, as well as a Defense Advanced Research Projects Agency (DARPA) Prophecy grant to R.A.

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Authors and Affiliations

  1. Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California, USA.,

    Ashley Acevedo & Raul Andino

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  1. Ashley Acevedo
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  2. Raul Andino
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Contributions

R.A. and A.A. conceived and designed the experiments; A.A. performed the experiments; and R.A. and A.A. wrote the manuscript.

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Correspondence to Ashley Acevedo or Raul Andino.

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The authors declare no competing financial interests.

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Acevedo, A., Andino, R. Library preparation for highly accurate population sequencing of RNA viruses. Nat Protoc 9, 1760–1769 (2014). https://doi.org/10.1038/nprot.2014.118

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