Data Analysis Support - Part 3
Reviewing the Sequence Peaks...
As explained above, each sequence result comprises two files:
- ".ab1" file: Chromatogram file containing sequence peak data
- ".seq" file: A simple text file containing the sequence
It is important when reviewing the data to look at both of these files. Viewing the sequence itself is easy, since any text viewer (Notepad, Wordpad, TextEdit, etc) will allow this. However, it is the ".ab1" file that gives access to the all important information that allows you to ascertain how accurate the sequence is. To view this file, you will need a chromatogram viewer. There are plenty available and lots of them are free.
When you open up a chromatogram file, you will expect to see coloured peaks of sequence data together with the actual sequence of the bases. That is what we would like you to see as well. However, that may not always be the case. Below are (simplified) descriptions of four types of chromatogram result you might see:
- "NNNNN". The worst case scenario. This means that there was nothing for the program to analyse. This usually indicates a "dead" reaction that failed to produce any fluorescent products capable of being detected. You should check that the template-primer combination is correct and double check that the template appears to be the right one. Basically do whatever you can to establish whether the reaction should have worked. Depending on what that shows, you may then want to contact us to discuss it further.
- Some peaks of fluorescence together with sequence. Peaks may be of poor definition, sequence may be short and there may be a high "background" signal as evidenced by additional peaks below and/or between the main peaks. This usually means that the signal intensity of the reaction was low and so the sequence quality will be poor. This can be confirmed by looking at the signal strengths (see below). You should check the same things as in (1), but also check the amount of template sent compared with our requirements. If the amount looks right, it might be that a contaminant is present that is inhibiting the reaction.
- Clean, well defined and evenly spaced peaks of fluorescence with corresponding sequence. Low / no background. Peaks continue like this for up to 1000 bases (assuming your template is that long). This represents a good reaction within the ideal intensity range that is generating high quality sequence.
- Initially, peaks with flat tops and/or additional peaks between certain main peaks. Often these extra peaks will be "read", thereby generating errors. Later on, clean peaks that generate high quality sequence. This usually indicates that the signal was too high and has resulted in "peak pull-up" near the start of the read. Due to the natural reduction in intensity as the sequence progresses, this effect decreases and gives way to clean, high quality sequence. Usually this is caused by there being too much template in the reaction.