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Dear Community,
Im seeking advice in whether there is a trustworthy and economical way to use NGS for highly targeted DNA methylation analysis (only 5 loci!), rather than doing RRBS.
We are interested on allele-specific methylation patterns within a cluster of imprinted genes in mouse. As far as I know, people usually purify DNA from hybrids derived from distantly related mouse strains, which thanks to their numerous polymorphisms, increase heterozygosity and allow distinction of the two alleles. DNA is bisulfite converted, then primers are used to target the region(s) of interest, the amplicon is cloned and multiple clones are sequenced by Sanger.
Our problem is that we want to analyze allele-specific methylation in five regions across multiple tissues of multiple animals. Due to the number of samples the experiment becomes very laborious and also very expensive. I am wondering if it would be possible to perform the same protocol up to the PCR but then take the amplified DNA and use them to prepare samples for DNA-seq where we can multiplex, can sequence rather shallow and maybe get all of our results in two lanes.
Because of the PCR there will be a massive number of PCR duplicates. In the classic protocol the PCR has 35 cycles and I’m not sure of how much lower I can go since PCR in BS-converted DNA is already pretty finicky. However, I wonder how much of a problem PCR duplicates will be, because in the end all I care about are the ratios of methylation between the two alleles, and the alleles should be equally represented. I will know whether this is true since all polymorphic bases should be 1:1.
I just have 2 questions: What problems am I overlooking with this approach? Are there other ways?
Sorry for the long explanation and thanks in advance for your help!!
Im seeking advice in whether there is a trustworthy and economical way to use NGS for highly targeted DNA methylation analysis (only 5 loci!), rather than doing RRBS.
We are interested on allele-specific methylation patterns within a cluster of imprinted genes in mouse. As far as I know, people usually purify DNA from hybrids derived from distantly related mouse strains, which thanks to their numerous polymorphisms, increase heterozygosity and allow distinction of the two alleles. DNA is bisulfite converted, then primers are used to target the region(s) of interest, the amplicon is cloned and multiple clones are sequenced by Sanger.
Our problem is that we want to analyze allele-specific methylation in five regions across multiple tissues of multiple animals. Due to the number of samples the experiment becomes very laborious and also very expensive. I am wondering if it would be possible to perform the same protocol up to the PCR but then take the amplified DNA and use them to prepare samples for DNA-seq where we can multiplex, can sequence rather shallow and maybe get all of our results in two lanes.
Because of the PCR there will be a massive number of PCR duplicates. In the classic protocol the PCR has 35 cycles and I’m not sure of how much lower I can go since PCR in BS-converted DNA is already pretty finicky. However, I wonder how much of a problem PCR duplicates will be, because in the end all I care about are the ratios of methylation between the two alleles, and the alleles should be equally represented. I will know whether this is true since all polymorphic bases should be 1:1.
I just have 2 questions: What problems am I overlooking with this approach? Are there other ways?
Sorry for the long explanation and thanks in advance for your help!!
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