Poly peak parser: Method and software for identification of unknown indels using sanger sequencing of polymerase chain reaction products.
Poly peak parser: Method and software for identification of unknown indels using sanger sequencing of polymerase chain reaction products.
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DOI：
10.1002/dvdy.24183
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发表时间：
2014-12
期刊：
影响因子：
--
通讯作者：
Yost HJ
中科院分区：
文献类型：
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作者：
Hill JT;Demarest BL;Bisgrove BW;Su YC;Smith M;Yost HJ
基因组编辑技术，包括ZFN、TALEN和CRISPR，已经产生了快速筛选许多F1个体以识别Indels携带者和确定突变序列的需要。目前的技术需要为每个个体对目标区域的多个克隆进行测序，这在必须分析许多个体时效率很低。直接Sanger测序是有效的，但由于错配区域，Sanger测序对插入序列杂合的基因组会产生一串“双峰”。为了便于Indel鉴定，我们开发了一个名为Poly Peak Parser的在线工具(可在http://yost.genetics.utah.edu/software.php)获得)，该工具能够将包含模糊碱基调用的色谱图数据分离为野生型和突变型等位基因序列。该工具允许根据直接在横跨靶部位的PCR产物上进行的每个个体的单次测序运行来确定插入序列的性质，而不需要克隆。这里描述的方法和算法有助于通过基因组编辑产生的杂合突变载体的快速鉴定和序列特征。虽然这个工具是为筛选F1个体而设计的，但在许多情况下也可以用来识别杂合子Indels。
Genome editing techniques, including ZFN, TALEN and CRISPR, have created a need to rapidly screen many F1 individuals to identify carriers of indels and determine the sequences of the mutations. Current techniques require multiple clones of the targeted region to be sequenced for each individual, which is inefficient when many individuals must be analyzed. Direct Sanger sequencing of a PCR amplified region surrounding the target site is efficient, but Sanger sequencing genomes heterozygous for an indel results in a string of “double peaks” due to the mismatched region. In order to facilitate indel identification, we developed an online tool called Poly Peak Parser (available at http://yost.genetics.utah.edu/software.php) that is able to separate chromatogram data containing ambiguous base calls into wild-type and mutant allele sequences. This tool allows the nature of the indel to be determined from a single sequencing run per individual performed directly on a PCR product spanning the targeted site, without cloning. The method and algorithm described here facilitate rapid identification and sequence characterization of heterozygous mutant carriers generated by genome editing. Although designed for screening F1 individuals, this tool can also be used to identify heterozygous indels in many contexts.