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Dev Dyn. 2014 Dec;243(12):1632-6. doi: 10.1002/dvdy.24183. Epub 2014 Sep 30.

Poly peak parser: Method and software for identification of unknown indels using sanger sequencing of polymerase chain reaction products.

Author information

1
Molecular Medicine Program and Department of Neurobiology & Anatomy, University of Utah School of Medicine, Salt Lake City, Utah.

Abstract

BACKGROUND:

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 polymerase chain reaction (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.

RESULTS:

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.

CONCLUSIONS:

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.

KEYWORDS:

Sanger sequencing; genome editing; indel identification

PMID:
25160973
PMCID:
PMC4525701
DOI:
10.1002/dvdy.24183
[Indexed for MEDLINE]
Free PMC Article

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