A flexible and efficient template format for circular consensus sequencing and SNP detection.
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.
Travers
Kevin J
KJ
Pacific Biosciences, Menlo Park, CA 94025, USA.
Chin
Chen-Shan
CS
Rank
David R
DR
Eid
John S
JS
Turner
Stephen W
SW
D001483:Base Sequence; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*; D013211:Staphylococcus aureus / Q000235:genetics; D013698:Templates, Genetic
D009841:Oligonucleotides; D004247:DNA