I am looking for a more technical answer, I see the standard illumina protocols suggest it, but can't find a clear explanation why. I assume it has something to do with standardizing reaction efficiency, but can it effect relative abundance of final taxa?
Abhijeet Singh
You don't have to agree with it but I'm just saying it really depends on where your DNA is coming from. If it is something like soil you will have a pretty diverse mix of bacterial, archaeal, fungal, algae, plant, etc. all in there and if you are only interested in say the fungal how do you know what proportion of each extraction is fungal. What is the point of normalization if you don't even know what proportion of your sample is what you are trying to normalization and how is that normalization even done?
What's your argument for standardization then? Sure you will have the same amount of input DNA but that is total DNA and could totally screw with the amounts of your target DNA which won't be normalized in either case and seems more like you are randomizing your target DNA concentrations based on total DNA amounts.
I suppose you are talking about the normalization your input DNA before the first PCR.
And I also hope you are aware of the amplification bias in PCR. Thus, normalization would help in the comparison of the cross sample data and also could be the other analysis with a bit of increased confidence.
The answer by Abhijeet Singh is correct that normalization would help in the comparison of the cross sample data. At the least, ensure that the DNA concentrations of your samples are similar (try to be in a range of 1-10 ng/ul) so that all samples have similar template complexity and the PCR conditions.
Hi,
to be a bit more precise. I am assuming you are talking about microbiome analysis as you refer to shifts in taxa. You should normalize your microbial DNA prior to the PCR as due to varying amounts of host DNA this can significantly affect your amplification. If you are interested I can give you more details on such an approach
Sven
I would also assume that in your case the gDNA you are using is more of less pure samples and are looking for variability in a "pure culture"? Compared to eDNA sample where normalization doesn't necessarily help much because when it is quantified you have no idea how much of the DNA is from your target organisms, so you may be unnecessarily diluting them and will not have an even starting point either way.
Michael A. Carson
" Compared to eDNA sample where normalization doesn't necessarily help much because when it is quantified you have no idea how much of the DNA is from your target organisms, so you may be unnecessarily diluting them and will not have an even starting point either way "
it not convincing.... at least for me... i dont agree with it
Abhijeet Singh
You don't have to agree with it but I'm just saying it really depends on where your DNA is coming from. If it is something like soil you will have a pretty diverse mix of bacterial, archaeal, fungal, algae, plant, etc. all in there and if you are only interested in say the fungal how do you know what proportion of each extraction is fungal. What is the point of normalization if you don't even know what proportion of your sample is what you are trying to normalization and how is that normalization even done?
What's your argument for standardization then? Sure you will have the same amount of input DNA but that is total DNA and could totally screw with the amounts of your target DNA which won't be normalized in either case and seems more like you are randomizing your target DNA concentrations based on total DNA amounts.
I think the only purpose for standardizing the amount of DNA to start PCR with is to help with amplification. If you have too little or too much DNA then you may not be able to amplify the DNA from your samples. Like Iryna said, you should have a similar amount in each reaction tube.
- Badges
- Science method