Reading chromatograms after bisulfite sequencing - Bisulfite sequencing (May/17/2005 )
I've been following various postings in the DNA methylation forum and found them to be very useful, particularly since am new with bisulfite sequencing and MSP.
So far, my bisulfite sequencing seem to be working well. I use Chemicon to bisulfite treat my DNA (DNA from mammary epithelial cell lines) and have been able to amplify a 435 bp product.
Thing is, when I get my sequencing results... the C residues at the CpG sites often come back as an N (unknown). Some have advised that it because of mixed alleles and that I should check the peaks of the chromatogram by eye and call it methylated if the peak is clearly C, unmethylated if it is clearly T and 'mixed' if I can see both a C and T peak. I suppose it is easy to tell if both the C and T peaks are of equal height, but how do I make a call if, say, the C peak, appears to be 20% of the height of the T peak? Would that still be a mixed? Also how can I tell if the 'mixed' peaks are not just background noises?
Am I the only one who finds this in my sequencing results, or does everyone get a clear C or T each time? Oh, I've also checked all the other non-CpG C residues in the sequence, and they are all Ts - which I presume means that the bisulfite treatment was complete.
Welcome to the methylation forum!
I think you are on the right track so far. You've got complete conversion because no C shows up at non CpG C spot.
OK, it's quite common to see overlapping C+T peaks. There is no clearly defined criteria to tell whether a lower C peak is a real one or just background noise. This has to be judged by looking at the overall background. If there are background C peaks of similar height, the overlapping C peaks may not be a true C. Generally if there is no much methylation in the sample, the background tends to be high. Sequencing using reverse primer usually yeilds better picture.
Thanks for your comments!
Ok, I'm glad to know that having overlapping C and T peaks are a common thing. Yes, I will double check by sequencing with the reverse primer.
I have heard that doing TA cloning also helps to give a more accurate picture. Is this so? Would you recommend it?
TA cloning will give clear cut data. At least 10 clones per samples have to be screened. Certainly you can screen more if cost is not a problem. If you are doing mechanism study I think cloning is a must.
I too welcome you to the methylation forum Labchick!
sounds like you are getting some nice results which is always good.
you can directly call the amount of methylation from the differing peak calls. I have seen people use a program called mutation scanner from amersham, that gives you a "percentage" for the SNP you sequence and has been used quite successfully for direct bisulfite sequencing.
cloning certaing gives you a cleaner result as pcrman suggests at least 10 to give an accurate picture and I would concur! the more clones you sequence the better the estimate of percentage methylation at one particular site.
Remember you are sampling the methylation profile of many many different cells with unique methylation patterns within your region of interest and you would expect to have variation in methylation levels at individual CG's with the exception of cancer cell lines.
good luck labchick!
Thanks heaps methylnick!
The samples that I have been working on are breast cancer cell lines, as well as a few immortalized mammary epithelial cell lines. Is it safe to assume that every cell from the same cell line will have identical methylation patterns?
Does methylation pattern change significantly with increasing number of passages? I've run out of DNA, so I've got to go back to growing the cells and extracting a new lot of DNA. Maybe this time, I can do TA cloning, and then sequence with forward and reverse primers.
Cells of a cell line may not have identical methylation pattern and that's why you got partial methylation at some CpG sites, although unequal methylation of two alleles may also appear as partial methylation.
Certainly methylation may change across passages, but if passages are close, it should be OK.
I agree with pcrman, cells within a cell line can vary in methylation pattern. The ideal situation is to look at the methylation pattern of a single cell, that would be a state of nirvana in the methylation field I would have to say. The closest thing to single cell bisulfite analysis that I have seen in the literature is something in the vicinity of 100 cells from memory.
There is a bit of confusion in the literature. One paper published in 1995 states that looking at methylation patterns in transformed cell lines could give rise to artefacts in that the genes that are not needed by the cell are switched off, therefore what you are observing is an effect of the cell line and not of the phenotype and this paper says you must look at primary lines. Another paper published by a Japanese group stated something along the line of, if the methylation pattern is established it will maintain the methylation pattern with high fidelity, this group looked at candidate promoters within a cell line over 100 passages by bisulfite sequencing and found very little variation in methylation across the number of passages which refutes the argument that methylation increases over time within a cell line. Certainly anedoctal evidence from my studies have shown that indeed the methylation pattern is established and maintained with high fidelity and genes that are not required by the cell line do not get switched off, ie: I have yet to see methylation of the promoters on our pet human chromosome sitting in a hybrid cell line.
In my case at least, I have yet to see the methylation change over time, as I have been looking at a region of interest and have run out of gDNA like you and had to reisolate more gDNA, of course I checked if the methylation pattern of one particular region was the same with the new DNA and it was!
good luck with it!