Hi
I perform targeted bisulfit sequencing of a specific gene from whole blood.
However, I get 3 different sequences in my results (when sequenced directly and also after cloning I get the same 3 sequences in my samples).
I have about 25 samples, after cloning I find 3 sequences in different samples and clones: the original bisulfit modified sequence of the gene (about 130bp) and 2 other sequences that are about the same length (one is +3bp the other -7bp) and hold both primers, but are nothing like the original sequence of the gene: the sequences start at the fwd-primer and ends at the rev-primer. They seem to be "hypermethylated" in so far that they have a lot of Cs in their sequence. Also, I have samples where I didnt find any clones with the "original" bisulfit treated sequence of the gene.
Just an example:
Original Sequence: AAGTCACGCG
Bisulfit treated original S.: AAGTCATGTG
Sequence 1: CACTCTAGC
Sequence 2: GGGTCTCAAAC
--> I find the last 3 (although all about 130bp, here just shorter as an example) in my samples
We have our theories, but are not "d'accord" in what is the best explenation, so I wanted to see what you think and if anyone maybe experienced the same thing?
Could these different sequences that I get with the gene-targeting primer be due to bisulfit treatment? Or is it really the problem that the primers bind somewhere else in the genome (although by blasting they were ok (of course in the untreated genome) and the sequences are all more or less the same length)?
Other explenations? Could allele-skewed methylation be an explenation for this?
So you treat with bisulfite first and then you amplify with PCR? Did you amplify both bisulfite treated and non-treated DNA?
May it be the case that the bisulfite treatment just converted some sequence to accidentally fit your primers? Especially if they are sequences of comparable size, you expect them to be more likely to be amplified to a similar degree in the specific PCR program that you used for your target sequence.
You did not get a BLAST match in your target organism. Did you BLAST the sequences to a broader database? You never know you have some contamination.
If the sequences have nothing to do with the target sequence, allele-skewed methylation seems unlikely to me, unless you are talking about some hyper-variable region where alleles may have drastically different sequences.
If the non-target sequence contains a lot of C that did not get converted to T, it is not heavily methylated, right?
Bisulfite modification process turns 4-bases (ATGC) into 3-bases (ATG) sequence except the 5mC. The sequence complexity is a lot lower and PCR mispriming is common. To get unique amplification, it requires unique PCR primer designs, PCR condition optimization, and PCR amplification validation to confirm the non-preferential amplification.
What PCR primer design software did you use to design the assay?
Hi,
Did you amplify any sequence to ensure that your DNA was properly bisulfite-converted?
I suggest the SNRPN promoter as control of bisulfite conversion. In normal conditions (exept patients with Pradel-Willi and Angelman syndromes) SNRPN promoter is methylated at manternal allele and demethylated at paternal allele. It serve as a good control for bisulfite conversion, particularly for allele-skewed methylation.
You can find the primer sequence here:
https://onlinelibrary.wiley.com/doi/epdf/10.1002/%28SICI%291096-8628%2819971219%2973%3A3%3C308%3A%3AAID-AJMG15%3E3.0.CO%3B2-N
Regards,
Yes, we include 4 controls (low, medium, high methylated DNA, and no DNA template) in each analysis starting from bisulfite modification. If Pyrosequenncing is used for bisulfite sequencing, every non-CpG C was monitored.
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