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Help with data from directly sequencing BSP product...


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#1 FFPE1

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Posted 20 May 2010 - 04:31 AM

Hi again!

Thanks for all of your help on previous posts! So I have been doing BSP and it is now working well for some of my genes of interest. I realise that cloning the BSP PCR products and then sequencing the clones is what many people do. But I see that some people directly sequence BSP PCR products (after they are cleaned up) and I would like to do this to try and save on time with cloning etc...

I sent BSP products for direct sequencing and I was wondering if anyone had some ideas/help with the results... So after BSP, I cleaned up my products using a PCR purification kit and I sent the PCR products for direct sequencing using the forward and reverse primers from the BSP PCR. To ensure it was working correctly I sent the amplified product using universally methylated or universally unmethylated DNA as template to be sequenced directly also. These results were as expected in that all C's were converted to T's except for CG's in the universally methylated samples in my region of interest. Therefore the bisulfite conversion worked well and the sequencing also worked well. I also sent a sample amplified from a cancer cell line and found all the CG's in my region of interest to remain as Cs. For all of the above the chromatogram gave a single peak for C or T in the CG dinucleotide regions of interest.

I also sent samples taken from the patients used in this study and found two peaks in the CG dinucleotide region of interest based on the chromatogram - one for C and another for T. Therefore, indictaing both methylated and unmethylated present. I realise that as I am directly sequencing BSP products I will have mixed populations in the sample sent for sequencing.... But does anyone have experience on how to quantify the methylation based on these results? Or any other help/tips in directly sequencing BSP products or analysing the data??
Thanks :)

#2 methylnick

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Posted 20 May 2010 - 01:18 PM

check your pm,

google is your friend.

there are many ways to analyse the data, strictly speaking it would be ideal to construct a standard with the sequence you are looking at with known spiked methylation and directly sequencing that.

But there are software that can analyse it for you. and example is the paper I sent to your pm.

Nick

#3 FFPE1

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Posted 23 May 2010 - 11:23 PM

check your pm,

google is your friend.

there are many ways to analyse the data, strictly speaking it would be ideal to construct a standard with the sequence you are looking at with known spiked methylation and directly sequencing that.

But there are software that can analyse it for you. and example is the paper I sent to your pm.

Nick



Thanks very much for your help!




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