hi,

in addition to what Maureen says you can get additional peaks from elecrical sparking from other equipment. Sometimes capillary sequencers have large backup batteries between the mains input and the machine in case of power cuts and these often minimise the sparking effect which would show in a reverse sequence but at a different position in the sequence.Failing capillaries can also have a poor effect on sequences and this would show between runs on the same capillary number..Nice sequencing though.

With a BLAST search of your sequence, I see that the site that is nearly 50:50 in your chromatogram is also a site that is often found as C or T in (and sometime reported as Y) in Globodera pallida 18S rRNA in GenBank (see attached JPG screen shot).  The other sites do not seem to be anywhere near 50:50 and are probably just a little "noise" in otherwise very clean looking data.  I don't know how many copies of the 18S rRNA gene you are expecting per genome, or if it is reasonable to find a 50:50 as you would for single copy gene in a diploid organism.  But this looks like sequence from such a case, and the database seems to confirm that other people have found the same result at this site.

The double bases indicated by a green arrow indicate that there are different ITS copies in the genome. This can be the result of a hybridization event (see attached file). The small peaks (red arrows) are most probably sequencing artefacts.

If you are looking for intra-individual polymorphisms, bear in mind that in a multicopy locus like the ITS it is difficult or impossible to detect a single polymorphic copy among the other copies that are present in the genome. Cloning the amplicon and sequencing multiple clones would be necessary.

The best way to resolve this problem is to repeat the PCR with high fidelity taq poly. I am a little bit concerned about the second and third red arrows from the left, because the second pic represent approximately 25% of the first pic and usually in sequencing, this % is considered. the other red arrows can be ignored. In my opinion repeat the PCR and sequencing of this sample after making a spatial calibration with the option "fill capillary" and then try to use another capillary position if you have a muticapillary sequencer (4,16...)..

To Marouane: your remark about the High Fidelity Pol is well thought. However, I did not mention it before (and maybe I should have), I already used a high-fidelity DNA Polymerase. Also my sequencing is done by a sequencing service, so not in my institute. I can also add that my products for sequencing were all purified from gel to avoid presence of primerdimers, left-overs from primers, additional unwanted PCR-products etc.

Indeed sometimes it is difficult to distinguish between 'noise' and an actual polymorphic site. Probably reverse sequencing (which I am doing right now), cloning (as suggested, but this means too much additional sequencing, probably I will select some samples to clone and see the results) and re-PCR (like you suggest) can help.

Thanks to all for replying and for the ideas.

As per chromatogram posted by you, i presume the problem persist might be due to the two reasons 

1) Bad spectral calibration of  dye set used in sequencing;

For this request your sequencer to re-calibrate the dye set (because always thymine [T] is showing over laps)

2) Due to (n-1) or (n-2) shorter sequencing primer;

Generally these kind of primers generates improper purification of oligos [use HPLC,RPLC or PAGE purified primers]

and/or

Degradation of primer during long term storage/multiple freez thawing/ storage in water