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

Library preparation for highly accurate population sequencing of RNA viruses.

Author information

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

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.

PMID:
24967624
PMCID:
PMC4418788
DOI:
10.1038/nprot.2014.118
[Indexed for MEDLINE]
Free PMC Article

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