Setting up bisulfite sequencing in lab - (Apr/10/2014 )

No, the reverse primer is for the COPY of the top strand which is made by the first cycle of extension with the forward primer. The bottom converted strand plays no role in this case.

You really are better off not thinking of the sequencing reaction as "PCR" as it is involves no exponential amplification and uses only a single primer. It's accidental that it happens in a cycler.

Hi again!

Well, thanks to all your help... I managed! I carried out some controls and it seems to have all worked out quite well! :) so thanks!

Now it comes to the data interpretation... as always, harder than the lab most of the times!

I have a question: I am analyzing my data using BiQanalyzer. I have just analyzed a trial sample I did, and analyzed my forward sequence, my reverse sequence (reverse complement) and it all aligned fine. Now when I got the print-out results it says that out of 13 CpG islands, my forward has 3 methylated ones and my reverse 1 methylated one. Would this count then that 4 out 13 CpGs are methylated (i.e. we just sum it up?).

thanks again!!

I don't use that program, and work almost entirely with bacterial cells, which don't methylate (always) at CG's. If your fwd and rev sequences align, then how can there be a different result for CG methylation in  one than in the other?

Mm yep, I am probably doing it wrong.

I sequenced with both the forward and reverse primers - I thought I had to align these to the original genomic sequence.

Therefore, I should align them amongst themselves and not to the original genomic sequence? Slightly confused on that issue.

The two sequences should be reverse complements of one another. First, verify that this is true. If not, then the sequence results are suspect (look at the electropherograms, check for dye blobs at about 80 bp and 120 bp from the start of sequencing, and for mis-calls). The single sequence you have can now be aligned to the genome, and this will tell you the presence of methylated C's.

Thanks!

sorted now. 

I have a doubt though - when I align my forward and reverse, what percentage of similarity should I be expecting? I'd like to in the future only do the forward sequencing (to save up on resources and time), but obviously have to be sure that the sequencing is correct.

If the DNA is pure and the sequencing good, then there should be 100% alignment. You are sequencing the upper and lower strands of a dsDNA molecule, and it should align. Impure DNA, low amounts, high amounts, bad priming, all could give poor sequencing results. You should be looking at the electropherograms to icheck the quality of your sequencing data. And, of course, reads longer than about 700 bp will start showing errors . Since you are working with short fragments, you may sequence off the end of the PCR fragment, and this gives weird sequencing results near the end of the fragment.