A normalization strategy applied to HiCEP (an AFLP-based expression profiling) analysis: Toward the strict alignment of valid fragments across electrophoretic patterns

Here we describe a method for the normalization of a set of time-course electrophoretic data to be compared. The method uses Gaussian curves fitted to the complex peak mixtures in each electropherogram. It searches for target ranges for which patterns are dissimilar to the other patterns (called "dissimilar ranges") and for references (a kind of mean or typical pattern) in the set of resultant approximate patterns. It then constructs the optimal normalized pattern whose correlation coefficient against the reference in the range achieves the highest value among various combinations of candidates. We applied the procedure to time-course electrophoretic data produced by HiCEP, an AFLP-based expression profiling method which can detect a slight expression change in DNA fragments. We obtained dissimilar ranges whose electrophoretic patterns were obviously different from the reference and as expected, most of the fragments in the detected ranges were short (< 100 bp). The normalized electrophoretic patterns also agreed well with reference patterns.The normalization strategy presented here demonstrates the importance of pre-processing before electrophoretic signal comparison, and we anticipate its usefulness especially for temporal expression analysis by the electrophoretic method.Amplified fragment length polymorphism (AFLP) is a DNA fingerprinting technique using electropherograms [1]. AFLP analysis belongs to the category of selective restriction fragment amplification techniques, which are based on the ligation of adapters to genomic restriction fragments followed by PCR-based amplification with adapter-specific primers [2]. This technique has been widely used for genotyping since it requires no prior knowledge of genomic DNA sequences and offers potentially better discriminatory power and speed than the existing techniques for fingerprinting such as random-amplified polymorphism DNA markers (RAPD) [3-8]. However, it has only been used to a limited extent for expressio