Accuracy of Automated DNA Sequencing: A Multi-Laboratory Comparison of Sequencing Results

摘要

A double-stranded (ds)DNA template of "unknown" sequence was distributed to approximately 80 core DNA sequencing laboratories by the Association of Biomolecular Resource Facilities (ABRF) for automated DNA sequence analysis. Forty-four different facilities responded with 8.3 usable sequence submissions. These sequences were grouped by both sequencing protocol (dye-primer or dye-terminator) and whether manually edited or not. The sequences were aligned with the known sequence, and the number of correct base calls, insertions, deletions, no-calls and miscalls were determined for each group. The dye-primer sequencing protocol provided the longest and most accurate sequence. The edited dye-primer data were >95% accurate out to 400--450 bp, while the edited dye-terminator data could call only 300-350 bases at this accuracy. However, 75% of the laboratories in this sampling preferred the dye-terminator protocol, presumably because of its versatility and convenience. Laboratories that manually edited theautomatically called data were able to obtain an additional 100 bases of good sequence when the dye-primer protocol was used. Surprisingly though, editing of dye-terminator results did not increase the amount of good se-quence, although the dye-terminator protocol had a superior base-calling ability within the first 100 bases of called sequence.