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Nucleic Acids Res. 2010 Aug;38(15):e159. doi: 10.1093/nar/gkq543. Epub 2010 Jun 22.

A flexible and efficient template format for circular consensus sequencing and SNP detection.

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  • 1Pacific Biosciences, Menlo Park, CA 94025, USA.

Abstract

A novel template design for single-molecule sequencing is introduced, a structure we refer to as a SMRTbell template. This structure consists of a double-stranded portion, containing the insert of interest, and a single-stranded hairpin loop on either end, which provides a site for primer binding. Structurally, this format resembles a linear double-stranded molecule, and yet it is topologically circular. When placed into a single-molecule sequencing reaction, the SMRTbell template format enables a consensus sequence to be obtained from multiple passes on a single molecule. Furthermore, this consensus sequence is obtained from both the sense and antisense strands of the insert region. In this article, we present a universal method for constructing these templates, as well as an application of their use. We demonstrate the generation of high-quality consensus accuracy from single molecules, as well as the use of SMRTbell templates in the identification of rare sequence variants.

PMID:
20571086
[PubMed - indexed for MEDLINE]
PMCID:
PMC2926623
Free PMC Article

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