DSDecodeM: An updated web tool for rapid decoding of multiple superimposed sequencing chromatograms

Sequence-specific nuclease-based genome targeting systems, i.e., TALENs and CRISPR/Cas9, often cause uniform biallelic and heterozygous mutations in diploid organisms. Direct sequencing of PCR products containing such mutations results in superimposed sequencing chromatograms. The Degenerate Sequence Decode (DSDecode) program serves to decode automatically sequencing chromatograms with biallelic, heterozygous, and homozygous mutations into allelic sequences.

A PC-version of this tool, DSDecodeML, is available upon requested.

For testing the program, five sequencing ab1 files and the corresponding reference are provided. Click download to obtain the test files.



Upload a reference sequence

Please enter an intact (wild-type) reference sequence of your target material.

It must completely cover the sequence range of the sequencing ab1 file(s) for one or multiple editting targets.

 

The program can search the two strands of the reference, so, it not necessary to convert the complement of the reference sequence.


Upload chromatogram files (up to 20 files)
 
Files
 
 
Note: to obtain high-quality sequencing files, it is better to use nested primers, but not the used PCR-primers, for sequencing.
 
The multiple chromotogram (*.ab1) files can be for the same target site or different targets coverred by the reference sequence.


Optional parameter setting

(Usually it is not needed to change the default settings and enter a target sequence)

Cutoff signal ratio (0.1-0.8)

Length of anchor sequence (10-20 nt)

Length of degenerate sequence (10-50 nt)

Target sequence of cleavage site (12-20 nt) *

* For decoding failed at first time, entering the target sequence would be helpful and the chromotogram (*.ab1) file(s) should belong to the same target.


Liu W., Xie X., Ma X., Li J., Chen J., and Liu Y.-G. 2015. DSDecode: A Web-based Tool for Decoding of Sequencing Chromatograms for Genotyping of Targeted Mutations. Mol. Plant (DOI: http://dx.doi.org/10.1016/j.molp.2015.05.009)

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