September 21, 2019  |  

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

Authors: Travers, Kevin J and Chin, Chen-Shan and Rank, David R and Eid, John S and Turner, Stephen W

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.

Journal: Nucleic acids research
DOI: 10.1093/nar/gkq543
Year: 2010

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