26 replies to this topic

[ConfusedAlice]

Hi everyone,

 

I am currently setting up the protocol for bisulfite sequencing from scratch in our lab, and I have a million questions (some maybe very silly..) but I am quite confused at the moment! I hope someone can help.

 

Up until bisulfite conversion I understand - extract genomic DNA and use appropriate kit for conversion (we have chosen Zymmo Research kit), with regular check-ups on quality/integrity, as well as use a control DNA.

 

But here the doubts start... we are going to use a 3130 genetic analyzer from Applied Biosciences as our sequencer. Now, although I have read and practised and tried all the primer design pages for bisulfite sequencing, I am confused as to what primers and PCRs to be carried out (or if just the One!).

 

Following my bisulfite conversion, is it possible to use the product obtained in a PCR with an appropriate polymerase/mix (we were thinking BigDye Terminator v3.1 Cycle Sequencing kit, Applied) and primers, and the end-product of this PCR use it directly for sequencing (after purification)?

 

Regarding my primers: I understand that the primers I get from places like Methprimer or Zymmo Research page are specific for bisuflite PCR, but since I have read so much about biotinylated primers, methylated specific, non-specific, etc. I am very confused as to which ones use and how many PCRs!

 

Any help or suggestions will be appreciated!!!

 

Thanks!!!!!

 

 

 

[phage434]

Here are the major steps:

1) Isolate genomic DNA

2) Convert part of that DNA using a bisulfite conversion kit (Zymo works well)

3) Design and order bisulfite specific PCR primers for the region of interest You should also order primers for the un-converted DNA for the same region.

4) Perform PCR on your converted DNA with the bisufite specific primers. Do PCR with the normal primers for the same region, on unconverted DNA.

5) Verify that a product was formed by running a gel. You should get the expected length band in both cases.

6) Take some of your purified PCR product and do a sequencing reaction. This involves mixing it with ONE of the two primers used in the PCR, and with the BigDye kit mix. Cycle, purify, and seqeunce. You should do this with both primers for each of the PCR products.

[ConfusedAlice]

Hi Phage434,

 

Thank you so much for your quick and helpful reply!

 

I have one question though regarding point 6) : when we use one of the two primers, you mean the bisulfite specific primers? So, we would use one bisulfite primer with our bisulfite-treated DNA, and the other bisulfite-primer with the unconverted DNA?

 

THanks again! 

[phage434]

No. You sequence each product in both directions. The bisulfite converted PCR product is sequenced with each of the forward and backward bisulfite primers; the normal PCR product is sequenced with each of the forward and backward normal primers. After conversion, it is just as if these are two completely separate samples. They have different sequences, and need different primers, both for PCR and for sequencing.

[pcrman]

How many samples are you going to analyze? My point is that if you don't have a lot of samples and many years of project to run, it is not worth setting up your own sequencing center since nowadays sequencing have become very cheap (~$5/sample)

 

As Phage has told, you will need a primer pair that only amplify bisulfite modified DNA to discriminate against unmodified DNA (for most conversion, there is likelihood of incomplete conversion). To do so, the primers need to be designed in regions which must contain a certain number of non-cpg Cs (e.g., at least 5), but do not contain any CpG Cs. If CpG Cs are unavoidable, you can use degenerate primers to cover both possibilities for the C, that is methylated (use a C) or unmethylated (use a T) C.

 

For bisulfite sequencing, the most difficult part is not at sequencing, but at PCR reaction! After PCR, there are two strategies for sequencing: direct sequencing and sequencing after cloning.  the former, although quick, cannot usually give very clean data (you may get high background/noise if PCR products are not clean enough); the latter gives your cleaner data, but labor intensive (you need to sequence at least 10 colonies).

 

If you have further questions, please let us know. 

[phage434]

Pcrman is correct. The most difficult part is design of good primers and getting the PCR reactions to work. Primer design can be handled these days by many programs. The pcr reaction is tricky because you are working with typically a degraded sample and the GC content is very low. The low GC requires a low annealing temperature, which most people expect, but it also requires a lower extension temperature, which many people do not understand. I routinely use a 64 extension temperature, and lengthen the extension a bit. This is a result of poor binding of the 3' end of partially amplified product to the opposite strand when the previous bases are almost entirely AT. You may also need to do a nested or semi-nested PCR reaction to get amplification.

[ConfusedAlice]

Many thanks Pcrman and phage434, I think I'm getting to it slowly! Excuse the late replies, but I like to read through it all to ensure I understand.

I have quite a few samples, but the idea in general is to set it up so our lab can use it routinely in the future.

Just to confirm concepts, once I sequence my two DNA's of one sample (one modified, the other not modified), I then align them so as to see where the mismatches are (I have been recommended a few software programs, such as BioEdit) and therefore methylation.

I also came across BiQ Analyzer (we need free software due to budget restrictions) - what is your opinion on these programs?

Thank you!

[pcrman]

For each sample, how many sequence regions are you going to examine?  

 

You don't need to sequence unmodified version of every sample. the purpose of sequencing a few of such sample is to let you know whether your modification procedure can successfully convert unmethylated cytosines. Comparing sequencing results from bisulfite modified DNA to reference sequences (from genome database) will tell you where cpgs are.

 

I have not used any analyzing program. I just use simple sequence alignment program to align the reads.  

[ConfusedAlice]

Hi again,

 

as it seems to be quite frequent from what I keep reading, I'm still stuck on the primer issue. I read that either the forward or the reverse has to be biotinylated, and I don't really understand :$

 

Also, I see some papers using pyrosequencing use a primer in only direction - why is that? I am doing "normal" direct sequencing, and don't think that would work for me, right?

 

million thanks!!!

 

A.

[phage434]

Some protocols may use biotinylated primers to purify the PCR product. These would not be the simplest protocols, and unless there is a good reason, i would not start with those.

 

All sequencing uses a single primer. I don't know anything about pyrosequencing that would make it different.

[ConfusedAlice]

Hi Phage434,

Thanks for your quick reply. I am now clear on the biotinylated issue!

I guess what still confuses me is:

- bisulfite specific primers: design according to certain criteria, forward and reverse.that part I think I understand OK.

- sequencing cycle: we use only one primer - forward or reverrse? Are these primers different from our bisulfite ones? This is the part I don't fully get.

Thank you!

[phage434]

For Sanger sequencing, it is common to perform two sequencing reactions on the same sample, one with a forward primer and a second with a reverse primer. This is done to catch potential sequencing issues. These primers can be the same as the PCR primers.

For next generation sequencing, it can be confusing, because often there is a requirement that very specific sequences be present at each end of the sequenced molecule. These are often added by adapter ligation, but can also be added as 5' extensions to a PCR primer. You really need to know which machine is being targeted, and it is a whole subject in and of itself.

[ConfusedAlice]

Ok great, I get it. We are using Sanger seq., therefore will use both directions. After one reaction we carry out the following, with or without some purifying before the second?

[ConfusedAlice]

Oops sorry sent too fast - just eanted to say thanks again!!

[phage434]

Typically the two Sanger sequencing reactions are carried out at the same time. After splitting the purified PCR product, each primer is added, and the sequencing reaction performed.

Both are not strictly necessary, depending on how precisely and reliably the results are needed.