Hi Vladislav,

I cannot be sure without seeing the original .abi sequence file which contains a lot of hidden information. Can you attach it please. My guess is that you have too much primer or too little template in your sequencing reaction and this means that you get a good strong signal in all of your bases up to about base 200. After that the intensity of the electropherogram drop to less than 10% of the early signal. I know that it does not look like this has happened but the software is taking the weak signal and pulling it up ( effectively changing the Y axis) so that you can see the problem. Unfortunately your analysis setting are set to ignore sequence below 10% of the early signal strength so it is only reading the very strongest later peaks like the Gs at 205 and 206 and ignoring the weaker ( perfectly good) sequence. If you re analyse this data with a lower cut off level for ignoring readable sequence then it will give you the full sequence.

Using the correct ratio of template dna to primer amount or using more sequencing mix will stop it from happening again but without the .abi file this is only my guess

I think it might be a program bug or some setting issue.

Looks like the "missing" data are for peaks that are below a baseline threshold of detection.

Dear Dr. Paul Rutland,

Thank you so much for your concern! Original .abi file attached to this message. I would be grateful for your advice.

Dear Junjie Shao , Katie A S Burnette , Laurence Stuart Dawkins-Hall

Thank you so much for the heads-up!

Dear Vladislav,

Thank you for the electropherogram. This looks like a different sequence but is very informative.

All of the running conditions on the 3130 are appropriate for a good run but the results are poor on this sample.

Firstly we see that the intensity of the base peaks vary from 114 to 146. This is too weak and indicates poor sequencing because they should be well over 500 so that the ever present background noise is insignificant compared to the signal intensity. As the intensity of the signal drops with sequence length the background becomes comparable to the signal and the sequence becomes unreadable. We can see also that at bases 25-40 and about 80 there are huge unincorporated dye peaks. This is important in 2 ways.

1 The dye peak is still large even though you have purified the reaction mixture so before purification there must have been huge dye peaks. This means that most of the sequencing reagents have not been incorporated into the sequenced product. This can be the result of 1a….old and inactive sequencing mix

2 1b too little dna template in the reaction.

3 1c too little sequencing primer leading to too little product

4 1d primer not annealing in the sequencing reaction. Unlikely as most people use 55c as an annealing temperature which is usually below the annealing temp of the pcr reaction

Situation 2

The large dye peaks are recognised by the analysis software and there is a background signal strength factor built into the software so it ignores analysis of minor peaks less than about 5% of the full strength peaks but if that 5% is relative to the huge dye peak then a lot of real sequence is less than 5% of the intensity of the dye peaks so will not be analysed.

If other researchers are getting good results from this sequencing kit then the kit is not a factor and you should check the amounts of primer and template in your reaction are as recommended in the BDT ver3 instruction booklet and possibly run a few extra cycles in the sequencing reaction.

Get back to me if you have any questions

Paul