Abstract
Automated fluorescent dye-terminator DNA Sequencing using capillary electrophoresis (a.k.a. CE or Sanger sequencing) has been instrumental in the detailed characterization of the human genome and is now widely used as gold standard method for verification of germline mutations. The primary information of the DNA sequencing process is the identification of the nucleotides and of possible sequence variants. A largely unexplored feature of fluorescent Sanger sequencing traces is the quantitative information embedded therein. With the growing need for quantifying somatic mutations in tumor tissue it is desirable to exploit the potential of the quantitative information obtained from sequencing traces. To this end, we have developed the ab1PeakReporter tool that converts Sanger sequencing trace files into comma separated value (.csv) files containing numerical data of peak data characteristics that can be explored and analyzed using conventional spreadsheet software. The web-based tool can be accessed after log-in into a user account at http://apps.lifetechnologies.com/ab1peakreporter. The output file contains the peak height and quality values for each nucleotide and peak height ratios for all 4 bases at any given locus allowing the detection and assessment of subtle changes at any given allele. We demonstrated the utility of this tool by analyzing samples with known amounts of spiked in variant alleles ranging from 2.5%, 5%, 7.5%, 10%, 15% and 25% and show that rare alleles could be convincingly detected around the 5%–7.5% level. In conclusion, enabling the high sensitivity detection of variants occurring at low level using Sanger sequencing will be useful as orthogonal verification method for next generation sequencing projects attempting to detect minor variants.