DSDecode： A Web-Based Tool for Decoding of Sequencing Chromatograms for Genotyping of Targeted Mutations

• Published in: Molecular plant 2015-09, Vol.8 (9), p.1431-1433
• Main Authors: Liu, Weizhi, Xie, Xianrong, Ma, Xingliang, Li, Jun, Chen, Jiehu, Liu, Yao-Guang
• Format: Article
• Language: English
• Subjects: Alleles; Computational Biology - methods; Genotype; Internet; Mutation - genetics; Sequence Analysis, DNA - methods; Software; Web; 图谱; 基因测序; 工具; 突变; 解码
• ISSN: 1674-2052; 1752-9867
• DOI: 10.1016/j.molp.2015.05.009

Dear Editor, The transcription activator-like effector nucleases （TALENs） and clustered regularly interspaced short palindromic repeats （CRISPR）/Cas9 genome editing systems have greatly improved the efficiency for generating targeted mutations in various organisms including plants （Li et al., 2012; Cong et al., 2013; Li et al., 2013; Feng et al., 2014; Ma et al., 2015b; Zhang et al., 2014, 2015）. In some plant species, the majority of mutations induced by TALENs and CRISPR/Cas9 systems are in uniform biallelic and heterozygous status in the first transgenic generation, although in some other plant species, chimeric mutations （with three or more allelic edited events within a single individual） may frequently occur （Li et al., 2013; Feng et al., 2014; Zhang et al., 2014, 2015; Ma et al., 2015b）. In many cases, it is necessary to determine the mutated sequences of the targeted alleles. However, direct sequencing （with the Sanger method） of PCR amplicons containing such biallelic or heterozygous mutations results in superimposed sequencing peaks starting from the mutation sites. Therefore, cloning of the mutation- containing amplicons and sequencing of multiple clones for each target editing site are required to determine the mutated sequences of the targeted alleles, which is tedious, time consuming, and expensive. Aimed at this problem, we have recently developed a highly reliable Degenerate Sequence De- coding （DSD） method （Ma et al., 2015a） and applied it to decode hundreds of targeted mutation events in rice and Arabidopsis （Ma et al., 2015b）. The DSD method decodes superimposed sequencing chromatograms in the following steps： （1） starting from the first overlapping-peak position on the chromatogram, manually generate a short degenerate sequence （DS） that is adjacent to the anchor sequence （AS）, which sits upstream of the first overlapping-peak; （2） query the DS against the intact reference sequence twice with a sequence analysis program to find the matched sequence（s）; and （3） link the AS with the query-matched sequences to generate the allele sequences or, if detecting only one matching hit, generate the second allele sequence by subtracting the allele 1 nucleotides from the degenerate bases. Even though simple and highly efficient, manual operation of this DSD method is still time consuming when decoding a large number of superimposed sequencing chromatograms.