Can we assume that this is sequencing of a PCR product and that there is one clean PCR product band on agarose gel. It would be useful to see one of the original sequencing files off the ABI machine as they contain more information. One possibility is that you have not completely removed all of the pcr primers before sequencing so this is a picture of 2 overlying sequences one from each end making it look messy. What method are you using to remove your primers please?. It is also possible to generate this effect with high background and low signal strength when sequencing too little product but your peak heights are good so I do not think this is likely. Can you post the .abi file and primer removal method please?

The lack of colours is worrying....it looks like the analysis of the sequence where the overlap between the dye colours has not been completed which would clean up the peaks greatly .It may be necessary to go back to the sequencer computer and re-analyse the data for the run. Check also that you are collecting all colours on the run...someone may have changed the data collection parameters but this is less likely after the start of a run

Dear Paul, I have like this problem in my segment, I used Ethanol precipitation of nucleic acids for purification and have low quality(low phred score) but I never used method for primer removal.if I remove primer improves quality of my segment!!!!!!

Hi Mahmoud,

it is essential to have only one primer present when you run the sequencing reaction or you end up with two sequences in the same electropherogram and it is unreadable. This is usually done by cutting the pcr band out of agarose gel or precipitation with PEG or some method which precipitates long DNA but not shorter primers or by destroying the single stranded pcr primers with exonuclease 1 in the exo-sap reaction. After one of these processes the sequencing primer is added to your template dna and the sequencing reaction is run. Have you had any good sequencing using your methods on other fragments before trying to sequence this fragment?

Thanks a lot Paul. Yes this is sequencing of a PCR product and there was also a clean sharp band for this product. The primer removal process used was ethanol precipitation and the sequence results found so far by using this method was good. 

I am assuming the same reason for the lack of colours in the peaks. How can I check the data collection parameters for any change? 

Here is the .abi files of this sequence run 

hi,I could not open the .abi file can you send it again or email me a file  please?.  Are other people getting good results off the sequencer with other templates or is everyone having problems. You can get this type of effect if the CCD in the sequencer is out of alignment but then everyone will be getting poor results. What are the conditions for precipitating the product but not the primes...I think that the likely problem is that  primer may be precipitating with the product and that you may be sequencing with two primers present in the mixture

Thanks and sorry for the inconvenience. I am sending you the .abi files through mail. Each run results this kind of peaks. No good results were obtained for last couple of runs. A test run was performed with the sequencing standard kit and got the same type of peaks. As there is no issue of primer precipitation or product purification in case of standard kit what will be the possible reasons? Will check the CCD alignment. Thanks again

Hi Paul,

Thanks for your guidance, I have five variety segment and had some bad sequencing and some another acceptable sequencing. in the best condition first 150-200bp have low phred score.  I only used forward primers for sequencing, my forward primer is not labeled however I do not know that Can I trust to exonuclease 1 or no?

why have some spots in sequencing high peak to anothers? and How I can decrease these peaks? 

I read manaul 3130 genetics analayzer and found if decrease concentration sample high peaks go down  but it was not effective.

Hi Mahmoud,

I find the exo/sap method excellent for almost all primers and very quick. I attach a simple method as commercial kits are expensive

EXO-SAP PROTOCOL                   

                                                                       for                          100 SAMPLES

EXONUCLEASE 1 ( 10U/UL)         (FINAL 2U/UL)         100 UL

SHRIMP ALK PHOS  (20U/UL)  FINAL 0.4 U/UL                       100     

10X SAP  DILUTION BUFFER                                          50

WATER                                                                                  250

 TOTAL                                                                                   500

SAP REACTION

 PCR REACTION                                            5-25 UL

EXO/SAP COCKTAIL                                 5

 MIX AND INCUBATE 37C  15-30 MINS

 INACTIVATE EXO/SAP  BY HEATING 80C FOR 15 MINS

NOTE EXO AND SAP BOTH ACTIVE IN PCR BUFFER

Add single sequencing primer to appropriate volume and run sequencing reaction.

it is excellent...try it

However Mahzabin above says that even the control sequencing sample in the kit is giving poor results so it is possible that there is a problem with either the run parameters or the gel or machine alignment  or even the analysis parameters.

Often though when you see a messy sequence degrading quickly there can be a problem with primer dimer generating a strong signal and using up all of the sequencing reagent . Also if primers can anneal to 2 places or there is some reverse primer present then the two sequences generated will look messy but when the control dna does not sequence it is possible that there is an equipment or analysis problem and sending a .abi file to applied biosystems technical help group has often been useful to me in the past

Maybe you have to perform a spatial calibration on the 3130 instrument. Someone might have openend the detection unit, and thereby moved the capillaries out of its position.

Other possibility is that the capilalliries are broken, so POP is present in your detection unit. This can happen if you tighten the capillary and the capilaries turn around and get crushed in the detection unit.

Hi Dear PAul,

I really appreciate it, Thank you for all your kidness.

I think it depends on the software you use to visualise the electropherograms. Sometimes certains softwares such as Sequencher de-colour the peaks when they are below a certain threshold of peak quality. Overall, the sequencing hasn't worked neatly.

Hi Martin,

Thanks. I have performed the spatial calibration after getting this type of peaks. And here is the outcome of spatial calibration. 

So now you performed a new calibrartion did you accept this calibration? If you accept this, the new settings will be applied in your next runs, so runs after this calibration should perform better.

Did you also look inside the detector wether there is no dirt inside, you can can use a N2 spray container to blow out any dust. 

You have to do a new spatial calibration after you have opened the detector.