Poly peak parser: Tool to identify indels from Sanger sequencing data

Prof H. Joseph Yost’s lab developed a software called Poly peak parser for identification of unknown indels using sanger sequencing of polymerase chain reaction products.This is a web-based tool to parse Sanger sequencing chromatograms with double peaks into wildtype and alternative allele sequences. Sequencing results parsed by this tool should have a region with single peaks followed by a region with double peaks. Sequencing results with more than two peaks, including those that occur due to multiple priming will not work. The single peak region is used to align the sequencing results to the provided reference to calculate the appropriate offset. The double peak region is then separated into wildtype and alternative allele sequences and aligned to each other. Both the alternative sequence and the alignment are returned to the user.

chromatogram

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. To facilitate indel identification, Yost lab developed an online tool called Poly Peak Parser  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.

Reference:

Hill JT, Demarest BL, Bisgrove BW, Su YC, Smith M, Yost HJ. (2014) Poly Peak Parser: Method and software for identification of unknown indels using Sanger Sequencing of PCR products. Developmental Dynamics.

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