Hello to all researchers. I'm new to the methylation field. This is my situation:
Questions:
Your input and information will be very helpful for me. Thank you very much in advance.
It is hard to say about the sequences without seeing the original .abi sequence files but your top sample has a long homo polymer run at the start of the sequence which depletes the sequencing kit of that base and leaves the signal intensity of the sequence very weak. It will probably sequence better from the other end.
Sample 2 has an enormous unincorporated dye peak suggesting that either the sequencing kit is going off or the primer is not annealing ( too little primer or wrong primer or too little dna template or that the purification of the sequencing reaction has failed as well as one of the above. It would be worth discussing this with the sequencing company
Dear Dr. Paul Rutland Thankyou for your insight. After read your opinion, i try to check the sequence from the other end (reverse direction), but it seem will end the same problem, i think because the sample is from bisulfite converted DNA, so there will probably be a lot of T. And for the second picture, the provider said the primer is anneal to multiple place. Do you have any experience with this kind of methylation sequencing procedure before that may usefull for me? Thankyou so much
hi Leonardo Tejo Gunawan ,
I have n experience of bisulphite sequencing but over 30 years of ordinary Sanger sequencing. Without seeing the original .abi sequence files it is difficult to be certain but I am not convinced that the primer is annealing in 2 places. They have to account for why there is a very strong unincorporated dye peak at the start of the sequence. This means that the sequencing reaction has not gone well but if the primers anneal in other places then there should be mixed (but strong) sequences and not much dye left. It seems more likely to me that the sequence intensity is weak and there is background noise being interpreted as sequence. Can you attach the .abi file please?.
I agree that there will be many Ts but not a whole lot of them at the start of the sequence so I was hoping that you would get more good sequence from the reverse primer before it hits the polyT
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