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direct sequencing - multiple sequences in BSP seq data

bsp sequencing

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#1 Lj Lee

Lj Lee

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Posted 12 August 2014 - 05:18 AM

Hi all,

 

I did a nested-PCR to amplify my template and I get a perfect band on agarose gel. Instead of cloning the PCR into different clones prior to sequencing, I sent my PCR products for direct sequencing. The template that I used are expected to be heterogenous as it was extracted from tissue samples. When I get the sequencing results, the data are not good, multiple sequences present in the read. I understand that this is due to more than 1 type of template in my PCR products but my question here is, is there any way for me to normalize this data so that it will be normal for me to compare the methylation rate of the same position? I'm new in this field and hopefully some of you can recommend a way or some software so that I can do the analysis. Or is it I must do the cloning for sure to avoid the noise?

 

Many thanks smile.png







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