Why is the Sequence in the Text File Slightly Different from that of the Chromatogram?

This page ONLY describes situations where the chromatogram and the text file agree MOST of the time, yet disagree in the first few nucleotides, or for individual nucleotides throughout.

If you see more than a few nucleotides difference between two supposedly-matching files (.Seq and .ab1), please contact the Core Director immediately. There's something quite out of the ordinary occurring!

Between early February and mid-March of 2001, there may be a few nucleotides difference between the sequence in your email (i.e. Seq files) and that found on the chromatogram. One of the two computers we use was misconfigured, such that any editing performed by the technician was applied only to the chromatogram. The .Seq file (and thus the text email) would remain as they were when they first came off the sequencer.

 

The data in all cases is your own data, and the differences between .Seq and chromat files were relatively minor.

Typically, a technician might edit a sequence in the following situations:

(i) the start point of the sequence wasn't ideal
Sometimes we can squeeze a few extra nucleotides of readable sequence out of the data for you by forcing the program to re-examine low peaks at the start of the chromatogram. You'd see 5-10 or sometimes even 20 extra nucleotides at the start of the chromat sequence, compared to the text file.
(ii) s/he felt the computer had made a mistake in calling the bases.
The basecaller gets confused sometimes, where a human can tell (with experience) what the nucleotide should be. We will sometimes manually correct problem basecalls. Again, if you see individual nucleotide differences occasionally, the chromatogram is usually the more accurate of the two sequences.

In each of these situations, edits performed by the technician would appear in the chromatogram, but not in the .seq (text) file. This means the data on the chromatogram may be slightly better, since the technician manually corrected problems.