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

A flexible and efficient template format for circular consensus sequencing and SNP detection.
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DOI:
10.1093/nar/gkq543
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发表时间:
2010-08
影响因子:
14.9
通讯作者:
Turner SW
Turner SW
中科院分区:
生物学2区
文献类型:
--
作者:
Travers KJ;Chin CS;Rank DR;Eid JS;Turner SW

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介绍了一种用于单分子测序的新型模板设计,我们将这种结构称为SMRTbell™模板。该结构由双链部分和单链发夹环组成,双链部分含有感兴趣的插入物,单链发夹环位于两端,为引物结合提供位点。在结构上,这种形式类似于线性双链分子,但它在拓扑学上是环状的。当置于单分子测序反应中时,SMRTbell模板格式使得能够从单个分子上的多次通过获得共有序列。此外,该共有序列从插入区的有义链和反义链获得。在这篇文章中,我们提出了一个通用的方法来构建这些模板,以及它们的使用的应用程序。我们证明了从单分子产生高质量的一致性准确性,以及在鉴定罕见序列变体中使用SMRTbell模板。
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.
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