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Setting up bisulfite sequencing in lab


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#16 ConfusedAlice

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Posted 03 May 2014 - 02:56 AM

Thanks for your invaluable help!

 

once I have sequenced in both directions separately I then realign each sequence (forward on one hand, reverse on the other) with the original sequence or amongst themselves? that part confuses me :$



#17 phage434

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Posted 03 May 2014 - 09:30 AM

Each forward/reverse sequence for the converted and unconverted DNA should align and (accounting for the reverse complement) be identical. As you gain more confidence in your sequencing ability, you may eliminate the reverse sequence, but for now I would do it.  The converted and unconverted should align except for the C's (or G's if you made primers for the reverse strand). C's that are present in the converted sequence started out as methylated. C's in the original sequence that appear as T's in the converted sequence were not methylated.



#18 ConfusedAlice

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Posted 06 May 2014 - 07:44 AM

Ok thank you very much! I think I'm having problems just to understand EXACTLY how the primers work - I want to understand everything perfectly! Will continuento read through the web to get the full picture, if you know of any reference which is a good read I would appreciate it!!! Thanks for all the help, it's verybappreciated!

#19 phage434

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Posted 06 May 2014 - 07:58 AM

An important starting point, if you really want to understand what is happening, is to realize that following conversion, the two strands of DNA which started out as reverse complements, are no longer complementary. The upper strand will have most of the C's replaced with T's. This also happens to the lower strand, but the sequence is reverse complemented, so when you look at it as normally written, most of the G's are converted to A's.

 

This means that when you are designing priimers, you are designing primers pairs for either the top or bottom strand, and those primer pairs are different. For the top strand, your forward primer will have mostly G's, and the reverse primer mostly C's. For the bottom strand, your forward primer will have mostly C's, and your reverse primer mostly G's.



#20 ConfusedAlice

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Posted 08 May 2014 - 12:20 AM

ok, so:

 

- when I use programs to design primers (i.e. Zymmo Research, etc..) the primer pair given is used for both the PCR following bisulfite conversion & the PCR cycle sequencing. The forward primer is for the top strand, and the reverse for the bottom strand?

 

- in theory, with only this primer pair I would have enough?

 

Thanks!



#21 phage434

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Posted 08 May 2014 - 05:12 AM

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.



#22 ConfusedAlice

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Posted 30 May 2014 - 07:08 AM

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!!



#23 phage434

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Posted 30 May 2014 - 11:17 AM

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?



#24 ConfusedAlice

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Posted 31 May 2014 - 02:54 AM

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.

#25 phage434

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Posted 31 May 2014 - 06:27 AM

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.



#26 ConfusedAlice

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Posted 04 June 2014 - 01:26 AM

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.



#27 phage434

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Posted 05 June 2014 - 06:18 AM

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.






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