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Despite intensive research efforts directed toward improvement of nucleotide dye labelling technology and modification of the polymerase used for automated DNA sequencing, the peak heights seen in sequence electropherogram data still vary significantly between positions within a sequence. This is largely because polymerase enzymes incorporate dye labelled di-deoxynucleotides into the extending DNA chain at variable rates during cycle sequencing. In consequence, heterozygous positions rarely contain peaks with equivalent heights; even when alternative alleles of a sequence are present in equal concentrations. In some cases one of the peaks may be so low as to be indistinguishable from background noise. This may result in inaccurate heterozygous base calls and has limited the applicability of DNA sequencing. We demonstrate that, even though peak heights vary considerably within a sequence, relative peak heights are highly reproducible between different samples when the same region is sequenced. We have used this approach to enhance base calling accuracy in our sequence based genotyping software package Assign-SBTTM. Sequence electropherogram data is adjusted as peaks of heights that are relative to the expected peak height. This has the effect of ΓÇ£normalisingΓÇ¥ the peak heights and results in electropherogram data where the homozygous peaks are of equivalent height and heterozygous positions of contain peaks approximately 50 of homozygous peaks. Base calling on this relative peak height data improves heterozygous base calling accuracy. This novel approach to sequence analysis combined with the unique and effective quality control analysis and high-throughput capabilities result in Assign-SBTTM being a comprehensive software package suitable for clinical and research re-sequencing applications that include genotyping, SNP detection, SNP scoring, pooled DNA comparisons and mutation detection and quantitation. David Sayer and Damian Goodridge Conexio Genomics, PO Box 1670, Applecross 6953, Western Australia www.conexio.iinet.net.au