How to interpret sequencing results? - (Oct/24/2006 )
I finally succeeded with direct sequencing thanks to (1) Qiagen columns, (2) Nick's PCR protocoll (excellent), and (3) CG enriched tag-primers .
I've gotten sequence information with both, forward and reverse primers . I've used the same template with different primer concentrations, so I was able to calculate correlations between the different conditions. A high correlation was present between the amount of methylation only for the reverse primers, but not for the forward primers. Forward and reverse primers do also not correlate .
Has anybody here gotten similar results? Is this, again, the mystery of forward primer sequencing in BSP? How can I be sure, which results are the "right" ones?
Many thanks in advance and for all the excellent comments in this thread, it really was helpful!
sounds like you have a nice little technical paper that could be published with regards to forward and reverse primer direct sequencing of bisulfite amplicons.
Could you explain what you mean by correlation between primers? is it higher concentration of primers you get higher methylation signals for your reverse primer and not so for the forward?
In a perfect world, the results you get from sequencing using the reverse primer should approximately be the same if not identical to the forward. It may not be the case because the base compositions of the forward and reverse are so different and skewed to three bases.
One way to overcome this is to use tagged primers and this has worked okay for me in the past. So to tag your primers with SP6 or T7 sequence and use them for direct sequencing.
thanks for your remarks.
I have already used tagged primers, adding C or G-rich tags in the second round PCR according to Han et al, 2006 (Pubmed ID: 16797472). In my opinion, this has led to the good forward sequencing results.
"Correlation" may be the wrong term - I just checked, if I aquire the same results in repeated measurements. Measurements of sequences using the reverse primer (in different concentrations) were highly reliable but sequences using forward primer were not. However, I just encountered that in some cases, forward and backward results are identical, so these measurements seem to be fine.
Just another short question: Somewhere in this thread it was proposed to score the direct sequencing results (like 0-20%=0; 21-40%=1, etc). Is this necessary or can you also use the raw values?
Hey, and btw thanks for your cycle conditions, that really helped a lot
Well you can't really use raw values unless you have run several calibrating sequencing reactions, where you artificially spike known amounts of the amplicon of interest with fully methylated templates against a known amount of unmethylated templates.
So by placing scores as into ranges of methylation level kinda takes into account the posibile variability from the PCR reactions, the sequencing reaction and the way the automated sequencer basecalls, taking note that the chromatographs we routinely see from a sequencing reaction has been normalised and processed.
I think the message to take home is to overt what you have done to score each CpG as I have found that most papers don't really state how a score was calculated.