In many large-scale sequencing laboratories, Phrap has become the sequence assembly program of choice. For optimal performance, Phrap requires accurate, base-specific quality values, which generally are generated by a companion program called Phred.

We have modified Base ImagIR, the LI-COR base-calling software, with the goal of generating trace files that are fully compatible with Phred and Phrap. The image processing parameters have been optimized so that Phred can generate accurate quality values from LI-COR trace files. Phred base calls generated from the optimized trace files give substantially lower error rates and significantly longer alignable read lengths, compared to sequences generated with previous versions of Base ImagIR software.

We also present a comparison of sequence qualities in projects generated entirely with LI-COR systems to projects completed with four-color fluorescent DNA sequencers. Higher accuracy and longer alignable read lengths were observed for sequence data generated on LI-COR systems that can potentially lead to better assemblies with fewer contigs, faster finishing, fewer mis-assembled repeats, and reduced costs.