Better DNA quality and quantity can definitely improve the Sanger data, that's the first thing I'd try.

Can you attach original sequence ( .abi) files from the company that did the sequencing please,They often contain more useful information than just an image of the electropherogram

Dear Amy Amy Klocko , thank you for your suggestion. I am wondering, whether nested PCR could reduce the effect of bad DNA quality? or enhance the problem?

Dear Paul Paul Rutland ,I have updated the question, now attached with the ab1 file. Thank you

Thank you Jeremiah Sia . The sequence can be roughly split into 3 sections,,,,bases 1-240, 240-580 and 580 to end

1-240 the peakintensities are over 1000 but the early quality is poor at the start but it is interesting that the underlying sequence is mainly G bases so I am thinking that the dna quality is good but either 1 the capillary on the 3500 is dirty and this is spoiling your results. Solution re run the sample on a different capillary

or the 3.1 kit is getting old and there is poor sequencing early on. This would improve with a new sequencing mix. Are there other good sequences with this sequencing kit. If so then I favour the dirty capillary possibility.

Bses 240-580 are good quality and good intensity ( above 250)

bases 590-750 are clean but there is low intensity signal and the peak separation is poor. You would get better results in this section by re analysing the sequence file with an equal spacing base caller instead of the kb basecaller but really the signal intensity is too low and again this could be caused by the sequencing mix being old or by too much primer or too little template dna

My feeling on your question to Amy is that nested or half nested pcr would improve signal intensity but if this is the only sample that looks poor quality then a dirty capillary could be to blame

ps you may be wondering why I quote such different signal intensities when they look so similar when printed. This is because the analysis software evens out peak heights to make it easier to read by eye but very often the higher number bases can be little different from background noise so the base call confidence can be low

Dear Paul Paul Rutland , this is valuable information, thank you so much! Well noted and i'll discuss with my supervisor for further actions. Maybe we'll try out sequencing services from different company to see if the problem persist. I typically received results like this for most of my samples. By the way, you mentioned that the bases from 1-240 comprised mainly G bases that it shows the DNA quality is good, may I ask how can I tell by observing the bases if the DNA quality is poor?

Hi Jeremiah,

I judge dna as good if the sequence signal is strong ( but not so strong as to use up all of the seq kit leading to signal fade after only a few hundred bases) which usually means too much dna or too much primer. There is no large multi base salt peak at 90 or 140 bases so the seq kit has been used in the sequencing reaction meaning that the unused reagents are easily and completely removed on reaction clean up ( a high salt peak inducates too little dna or primer or a failed seq reaction. The backgroung noise on the electropherogram is also low so the signal to noise ration is high which usually means good quality dna being sequenced

Dear Paul Paul Rutland , understood. Thanks a lot for the explanation.

Dear Jeremiah Sia,

I came across all your results that your attached the images of electropherogram and .ab1 file was clearly explicit the problem. I have worked on the sanger sequencing machine closely and the main problems that can interfere your results are 1. Your Quality of DNA ( Your saying your confident and that you mentioned it is 1.4 to 1.7) But most of the cases we expect the quality 1.7 to 1.9. Further, you should ideally provide at least 10 ng/uL as a starting concentration for the downstream in order obtain good results. I hope you would attach a gel image for clear understanding. 2. I totally agree with you that you have used the nested PCR to reduce the non specific amplification I expect there will not be much contamination with your PCR product. I would expect perhaps you obtained a good quality of gel image post PCR. 3. PCR Purification Kit I hope the buffers you are using free from accumulation of salts, that will sometime clog the capillaries during sequencing and that will effect the Q.C of your results. 4.The outsourcing company reagents, there is called dilution buffer that some companies for their benefit they will go up to least dilution. So, you should ensure the dilution they are using for your product. 5. I hope you provided requested volume of your product along with your forward or reverse primer if they mention. These all are technical points I can assure you it saves most of your time and avoid lot of confusion later on.

Dear Srinivas Srinivas Chaganti , thank you for your explanation. Well noted and I will also try to improve my DNA extraction protocol so i dont have to do nested PCR for every sample. i suppose the low 260/230 reading is due to the DNA extraction kit that i am using, which contains EDTA. Due to my low DNA concentration, I had to increase my eluted DNA mixture volume to 10uL during PCR, which i suppose could also mess up with the sequencing slightly?

Note:

1. the ab1. file i attached in the question is post-nested PCR

2. i attached here the DNA gel electrophoresis image for few of my samples (these are better ones) and the electropherogram ab1. file of the same sample and same region without nested PCR

Dear Jeremiah,

Exactly you are on the right track to tackle the situation. Yeah hope now you are troubleshooting the issue. The DNA Extraction is the major issue in your case. I hope if you can optimize accordingly there will not be any issues further for downstreaming. Although playing with fungus is always a tough job I agree and extraction of genetic material will also play a crucial role. Follow some available standards that make your job so easy. I wish you all the success in your future endeavors.

Dear Srivinvas, thanks!

The first picture (P3) looks okay. But the third picture (P2) of chromatogram shows noise. It's normal to get noise till first 30-40 base pairs but after that the peaks become sharp and clean.

You can get noise due to number of reasons like : 1. if the DNA extraction was not done properly. 2. If you have primer dimer bands after PCR.

3. if the sequencing primers were contaminated somehow.

Hi Jeremiah,

When you say that your sample concentration is low, what is the length of your PCR product, and what is the actual concentration? Our Facility typically asks for a concentration between 2.5 ng to .5 ng per 100 bases.

Are you submitting your samples in water or TE? We recommend water because high salt concentrations can cause issues with sequencing.

Do you have access to a Qubit? A nanodrop can give you false concentration readings.

Have you tried using Amersham's ExoSAP-IT kit or a Microcon filter for PCR cleanup? If your PCR results yield multiple bands or primer dimers, you may want to try Qiagen's QIAquick gel extraction kit.

What is the Tm of your primers? PCR primers do not always make good sequencing primers. Sequencing primers should be at least 18-20 nucleotides in length with a melting temperature of around 52-55 °C (calculated by the formula Tm = 2(A+T) + 4(G+C)). If the Tm is higher than 60, you might want to let your sequencing core facility know.

Have you considered cloning your PCR product into a plasmid?

Finally, I would probably see if the core facility you use offers a GTP premix or a mixture of standard Big Dye and GTP mix. GTP mix substitutes dGTP for the dITP found in standard BigDye. Using GTP, a mix of GTP and Big Dye, and/or betaine in the sequencing reaction mix can help alleviate problems due to secondary structure.

I hope these suggestions help. Good luck.

Dear Saileyee Saileyee Roychowdhury , thank you! i will try to improve my DNA extraction as well. Occasionally i do get primer dimer. I am wondering, we used sterile dH2O instead of ddH20 to dissolve the primers and also in PCR, would that be a cause for contamination?

Dear Kevin Kevin Cavallin , thanks!

My targeted region is about 980bp but I am not sure about my PCR product concentration, we sent the PCR products out to sequencing company directly after PCR. They will do the cleanup and sequencing.

I did my elution during DNA extraction using TE buffer. Thrugh nanodrop, I generally get low DNA concentration after extraction, its usually lower than 5ng/uL, occassionally im able to get 8 or 10ng/uL. Due to low DNA concentration, I used more of the eluted DNA mixture (with TE buffer) during PCR, usually 10uL.

We have a Qubit in the lab, I'll give it a try!

The Tm for the primer pair of this sample is about 61 degree celcius

Regarding cloning, I was suggested of this option, I may give it a try.

The sequencing company used standard BigDye, but i'll confirm with them if they had included GTP mix.

It is always helpful to know more about where things could go wrong

Dear Jeremiah,

Please dissolve your primers in autoclaved MQ prepared from ddH2O or in TE buffer. Also, if you are getting primer dimers after PCR, then please ask the company to perform PCR clean up before performing Sanger sequencing. This step might improve your results.

Good luck with your experiments!

Dear Saileyee Saileyee Roychowdhury , well noted and thanks for your kind wish!

For a PCR product that is 980 bp, we recommend a concentration (post-PCR clean-up) between 0.0245 ug/ul and 0.0049 ug/ul (We run a 10 ul reaction). We recommend submitting samples in biotech water versus TE because salts and EDTA can cause problems. With a Tm of 61 degrees, I would run a 60-degree annealing/extension versus the standard 50-degree annealing and 60-degree extension when doing the sequencing reaction. Finally, I would use a 1:1 mixture of standard BigDye and GTP mix. That would be in addition to using Betaine or DMSO. I hope these suggestions help. Good luck!

Dear Kevin Kevin Cavallin , thanks very much on your suggestions! i'll try to mention your suggestions to the sequencing company.

Dear all, I tried out sequencing with another company, turned out the sequencing part was really the problem. I finally got clean looking sequences now. Thank you all for your responses, it has been really helpful to me, especially to understand what are the potential causes to the problem could be. May God bless you all.