Dear Masoomeh

Thank you for the .abi file

There are some interesting features. First your products are good but in the first 6 samples there is primer dimer that will badly effect sequencing because although the signal on the gel is weak the dimer is short so binds little dye so actually there is a lot of it and it will bind sequencing primer and give short sequences and the reagents will run out early in sequencing. We have to trust that the purification method pre sequencing removes all primer dimer.

Overall the signal strength of the sequencing of your sample is very weak. Base signal strengths 57-85 and background signal strength of 7-9 RFU. This means it is almost impossible to distinguish signal from background so the sequence is unreadable. The analysis parameters are for BD3 and the sequencing consumables are in date.

If this were all of the information then possible reasons for poor signal are…too much salt in the sample, too much sample in the sequencing reaction,too little primer in the sequencing reaction but the last 2 of these would give you some good sequence but no signal at the longer reads. This is not the case…your signal is uniformly low.

Your 3730xl sequencer is running a 50cm capillary array for which the sample loading conditions should be between 15seconds and 45 seconds at a loading voltage of 1500 volts ( higher is allowable in certain conditions)

Your .abi file shows that the samples were loaded for 3 seconds at 800 volts. I think that these loading conditions means that very approximately one tenth of the usual amount of sequence reaction has been loaded and this means that the signal strength is too low to analyse. I think that the sequencing is probably fine and that if these samples wee run again at the usual loading parameters that they would look good.

I attach the informationbooklet about this sequencer for your interest

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Hi, can be many reasons behind.

how are you doing PCR purification? Maybe concentration is low, did you measure it?

Hi!

It seems like your sample got contaminated. What is your experiment design? Are you sequencing single colony?

Best regards!

Tong

Dear Masoomeh Rajabi,

Based on your chromatogram picture, it could be your DNA sequencing process is failed. It shows from the unclear and overlapping of your sequence peak moreover in the length of ~330 bp position.

This may be caused by clone contamination or low purity of your PCR products.

I attach the troubleshooting file for the interpretation of your sequencing result. Hope it’s useful.

Regards

Dear Diana

Thanks for your answer

I didn't PCR purification and my samples are plant. Sequencing company has done purification

Dear Tong

Thanks for your answer.

I don't have colony. my samples are plant and I used some DNA bar coding primers.

I have also done sequencing using the same barcode primer (rbcl) and my first suggestion would be to optimize the PCR condition, it seems that you probably had some extra bands or smearing of the sample..since you sent the sample somewhere else, i am guessing that the cleanup process was not done by you. Nevertheless, there are several possibility for such noisy chromatogram, either you had cross contamination between the plant species or there were some non-specific bands during the PCR.If possible could you please upload the picture of the band as well? However, it is without a doubt that sometimes such things happen and could be the problem of the sequencer as well. For starters, you could try to perform PCR with HF taq polymerase and also decrease the concentration of the Magnesium chloride in the PCR mix, which should give you error free PCR amplicon upto certain point, but as the length of rbcl is relatively less, these might not have been in the problem in the first place, but still you could try optimizing the PCR condition so as to have a clean band. Finally, from the look of things, the only viable option for you is to re-send the samples for sequencing and hope that all goes well..

Dear Mitesh

thank you so much for your answer. I don't have any nonspecific band. the picture of pcr is attached.

I don't understand what you mean in this sentence( cross contamination between the plant species ).

When I said cross contamination, I meant may be during extraction the plant samples got mixed up accidentally. It would be helpful if you could point out the band from which the above sequence chromatogram was obtained and also the ladder information.

my ladder was 1Kb and unfortunately all of this bands have

Chromatograms like the image that I attached.

If the quality of DNA may lead to such problem in Sanger seq?

Hi Masoomeh,

Those gel bands are lovely and you should get good sequences back from them though you do have non-specific bands, they are faint and should not result in the chromatogram you uploaded. You could probably remove them by shortening the Ta time.

Is your chromatogram that noisy from the beginning? As it suggests more than one nucleotide is being detected at a time which would indicate contamination.

Have you checked your primers are appropriate for sequencing? There are parameters for sanger sequencing. Have they been used before, successfully?

Have you tried sequencing with the reverse primer?

You could gel purify one or two bands and send them for sequencing too. This would be very informative as a gel purified fragment won't contain non-specific PCR product. If that doesn't give you a clean chromatogram then it may be your primers.

Let us know how it goes,

Best wishes,

Daniel

In your case, test to analyze your sequence data without seeing the chromatogram first in NCBI blast. If you got the results showing the plant species that appropriate with your wish, at least it’s already give you a short answer.

Later, try to re-send your samples (pure PCR products not PCR products) for sequencing. If you still get the same chromatogram, it could be your primers or just technical error in doing PCR sequencing or purification of PCR sequencing.

Your PCR bands looks good. It should give you good sequences as Mr. Daniel Brady said to you.

Good luck!

can you attach the original .abi file please.There is much more information in the abi sequence file than just the electropherogram

Hi @Masoomeh, all the bands from the photo are with the same primer? Usually companies ask to send PCR products in a certain range of concentration, when they do the purification they will ask more concentration than when the purification is already done by the scientist. So, I believe too few concentration or to high is not good for sequencing reactions. So, check what concentration range is asking the company and confirm the concentration of your products is in that range.

Other option is to use an internal primer for sequencing, so this primer should give you better results for sequencing in case your other primer is not specific enough, but i do agree that the bands you have seem to be good enough. Godd luck

I've seen that sort of "noisy" chromatogram when you don't start with enough PCR product for the sequencing reaction and/or when you have more than one type of DNA molecule in your PCR reaction.

Dear Rajabi

I think that either you use low concentration of DNA or a high chance of contamination occurred. Also, You could gel purify one or two bands and send them for sequencing other time....Regard

Thank you all so much for your nice answers

I attached the ab file

I think either there is more than one copy of the targeted loci in the PCR reaction, or you got DNA carryover! Pay attention to these fading double bands!

Best luck

Dear Masoomeh

Thank you for the .abi file

There are some interesting features. First your products are good but in the first 6 samples there is primer dimer that will badly effect sequencing because although the signal on the gel is weak the dimer is short so binds little dye so actually there is a lot of it and it will bind sequencing primer and give short sequences and the reagents will run out early in sequencing. We have to trust that the purification method pre sequencing removes all primer dimer.

Overall the signal strength of the sequencing of your sample is very weak. Base signal strengths 57-85 and background signal strength of 7-9 RFU. This means it is almost impossible to distinguish signal from background so the sequence is unreadable. The analysis parameters are for BD3 and the sequencing consumables are in date.

If this were all of the information then possible reasons for poor signal are…too much salt in the sample, too much sample in the sequencing reaction,too little primer in the sequencing reaction but the last 2 of these would give you some good sequence but no signal at the longer reads. This is not the case…your signal is uniformly low.

Your 3730xl sequencer is running a 50cm capillary array for which the sample loading conditions should be between 15seconds and 45 seconds at a loading voltage of 1500 volts ( higher is allowable in certain conditions)

Your .abi file shows that the samples were loaded for 3 seconds at 800 volts. I think that these loading conditions means that very approximately one tenth of the usual amount of sequence reaction has been loaded and this means that the signal strength is too low to analyse. I think that the sequencing is probably fine and that if these samples wee run again at the usual loading parameters that they would look good.

I attach the informationbooklet about this sequencer for your interest

�~

You have to re- purification and check the concentration may be low

regards