Noisy "C" signal in the sequence - (Feb/15/2006 )
Dear All,
I use PCR product as the template for bisulfite-treated DNA methylation test. Some of the sequence show "noisy" signals which is not an actual "C" signals but mixed in the sequence results. Because they don't show in all the sequences and I noticed that such results were more likely to show in those complete converted "C" sequences, is there anyone had such problems before? How to overcome it? Thanks
hi mou1,
I was wondering if you could clarify your problem a little, are you performing direct sequencing of PCR products? or are these coming up in clones you have sequenced?
It would certainly be good if you are able to post an image up for us all to see and comment on.
Cheers
Nick
Fantastic! Someone with the same problem as me.
'noisy Cs' are blue C peaks appearing all over the place in the chromatogram, where there weren't even Cs before bisulphite treatment.
Mine are from direct sequencing of PCR products. (My old boss published papers where relative peak heights of C/T in CpGs were calculated from direct sequencing rather than using the cloning method).
This has been addressed before. With direct sequencing, there is a deficiency on C signal or G signal depending on the orientation of your sequencing reaction (check the fluorescence values for the C or G signals, they would be at least 10 fold out compared with the values of the other three bases).
The noise you are seeing could be from the baseecaller overcompensating on the C signal and normalising with the other signals......with direct sequencing there will always be a residual background C signal because there will always be unconverted template within the PCR amplification and this can be picked up by direct sequencing.
I will show you an example below of background C signals, this is usually what I get.
Nick
Thanks Nick.
There are two reasons that I can get from the reply. One is the unconverted 'C". For this reason, I carried out TA clone(10 clones), and there is no "C" signals anymore. The second reason, is the compensation. Because I use 377 sequencer to analyze results by myself, I wonder is there any way to block those automatic compensation. Thanks.
with the ABI basecaller, this is a known problem and can not be rectified easily the last time I spoke to ABI. You should approach them yourself as the more people who complain about the problem, the more likely they may do something about it.
Nick
Any ideas why the basecaller overcompensates only some of the time? Some of my sequencing looks really good.
Thanks,
Debbie
another reason could also be imcomplete conversion and there are some amplicons that have C's in them....that is another reason....as for why the basecaller is doing that, you would have to ask ABI.
Nick
Hello all,
I had the same problem with direct sequencing of my bisulphite PCR products, especially when they are totally unmethylated and one base is completely missing (in this case, it usually works better the reverse primer to direct sequence). I reported the problem to ABI and it seems like there is nothing to overcome the problem. However, someone told me about a basecaller that overcomes this: KBTM BASECALLER (improved analysis of samples with very skewed base composition).
Does anyone know anything about this basecaller?
Good luck to everyone doing direct sequencing of bisulphite PCR products 
Cris
direct sequencing always works better with the reverse primer, I have seen this also.
as for the base caller, the KB™ Basecaller is the one that comes with every ABI sequencer, maybe ther most recent revision of this software can cope with skewed base composition samples?
Nick