direct bisulfite sequencing - (Jul/10/2007 )
I'm recently learning bisulfite PCR and would like to directly sequence my products. Now I'm not sure how many times I should sequence a sample from one individual. Should I repeat the whole procedure (from bisulfite modification to sequencing) more than one time?
Any help is greatly appreciated, thank you.
Well I gues there are a lot of different answers to this question.
I am currently working on rat brain and I do the following: bisulfite in duplicate, PCR amplification without duplicates, sequencing duplicates or triplicates, leading to 4-6 traces per sample.
I am currently working on rat brain and I do the following: bisulfite in duplicate, PCR amplification without duplicates, sequencing duplicates or triplicates, leading to 4-6 traces per sample.
Thank you krümelmonster!
I have also some problems with the interpretation of the sequencing results. I know that I can obtain two peaks at one site (c/t). Is it a result of incomplete bisulfite modification?
The height of C peak relative to T peak is a methylation level. I'm not sure what it exactly means.
You'Re almost there Agatha! First, I would recommend you concnetrate on the Reverse Sequencing, as this normally works better than Forward.
When you have your traces, your first look should be at the non-CpG C's. Those should all be converted to T's. Remaining C's indicate incomplete conversion. Next step - analyzing the CpG-C's. After aligning the sequence, you search the CpG sites in your sequence. There you should have to peaks (C/T or G/A respectively). You can roughly quantify the amount of methylation by the formula: %methylated C = 100 * peak height C/(peak height C + peak height T). This tells you if there are methylated sequences in your sample.
When you have your traces, your first look should be at the non-CpG C's. Those should all be converted to T's. Remaining C's indicate incomplete conversion. Next step - analyzing the CpG-C's. After aligning the sequence, you search the CpG sites in your sequence. There you should have to peaks (C/T or G/A respectively). You can roughly quantify the amount of methylation by the formula: %methylated C = 100 * peak height C/(peak height C + peak height T). This tells you if there are methylated sequences in your sample.
So, I look first at the non-CpG C's to be sure that conversion is complete. Then I measure peaks height. (Is there any special program for measuring peak height?) Finally I obtain %methylated C in my sample at one particular site and I still don't understand...
Previously I thought that the term "methylation level" concerns the whole CpG island region, but using the formula %methylated C = 100 * peak height C/(peak height C + peak height T) I can calculate methylation level of one CG. From my point of view one CG can be methylated or not. So, why can I obtain such a result of direct sequencing - two peaks at one site?
Maybe because in my sample I have a mixture of cells which are differently methylated?
My questions must be very silly for you, but I'm at the very beginning with methylation and such techniques as bisulfite sequencing
thank you for your help!
You can only determine the level of methylation directy for one single CpG site. Analysis of all CpGs within a CpG island will then provide you with a greater view of the methylation level of the island. But keep in mind that very few, even single methylated CpGs are at times sufficient to epigenetically alter the expression of a gene!
You can get mixed signals because (as you mentioned) you have a mixture of different cells altogether, or because of allelic differences in methylation within a single cell. Example: 2nd female X chromosome is methylated. When looking at methylation of x-chromosomal genes in a female cell, you would expect a C and a T peak in the forward strand.
But here's my question, Krümel 
:
I often get nice clean signals in both the forward and the reverse strand, but the reverse strand tends to show a lot less methylation, actually giving me different results when comparing it to the forward strand from the exact same sample. It seems the reverse primed strand is less sensitive to methlyation, I can't make any sense of it.
Also, the reverse strand's signal is a lot weaker and basically has no background noise at all.
The forward strand has a far greater peak heights and considerably more noise, although the noise is well within tolerable bounds. It shows clean and clear double C-T peaks where the reverse shows a clean single A peak.
Any insight?
That is really interesting, cyburn, as it is normally the other way round for me! I have no good explanation for it, sorry.