Library preparation for highly accurate population sequencing of RNA viruses

Nat Protoc. 2014 Jul;9(7):1760-9. doi: 10.1038/nprot.2014.118. Epub 2014 Jun 26.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Algorithms
  • Gene Library*
  • Genetic Variation
  • RNA Viruses / genetics*
  • RNA, Viral / chemistry*
  • Sequence Analysis, RNA / methods*
  • Tandem Repeat Sequences

Substances

  • RNA, Viral