The question of accuracy and length of read in DNA sequencing was addressed recently by Koop, et al. (Biotechniques 14:442, 1993). Sequenase was used for manual sequencing in 96-well microplates and Taq cycle sequencing was automated. Approximately equivalent results were obtained for both sets of data involving a total of 773 sequencer runs. The observed error rate was about 1% over the first 350 nucleotides but increased to 17% at 500 nucleotides. Deletions, mismatches, ambiguities and insertions were all well below 1% for both methods through the 350 nucleotides. Deletions peaked at 400-450 nucleotides. Mismatches and ambiguities increased to more than 7% at 500-550 nucleotides. Error rate due to insertions began increasing after 460 nucleotides.
Between nucleotides 350-450 the major source of error was deletions. In the region of 450-550 nucleotides the major source of sequence error was mismatch in Taq cycle sequencing and both mismatch and ambiguities in Sequenase sequencing. Neither method demonstrated a significant advantage in terms of providing longer runs.
A primary consideration regarding the choice of method is the amount of template needed; Taq cycle sequencing requires about one-third the amount necessary for manual Sequenase reactions. While their automated method required more time to complete a set of reactions, consistency and throughput were considered overriding factors.
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