Hello,
I'm interested in quantifying the number of bacterias in various samples (from fly microbiota), in order to compare the effects of 2 treatments. One treatment may diminish the abundance of bacterias compared to the other.
I generated OTU tables from the results of a MiSeq analysis, and I was wondering if I can use the number of reads as a way to compare the abundance of the OTU between my treatments.
I know that I can easily perform relative abundance of the OTU intra-sample, but I would like to do it inter-sample (and never saw this in the litterature).
Since I extract total DNA (fly dna + bacterial dna) should I normalize the quantity of DNA before my pcr ? Any ideas ?
Thanks
Bonjour!
Maybe you can do the relative abundance of the OTU or OTUs comparing with the fly number of DNA sequences (if you have any of them in the Miseq reads), You can't use only the number or reads because the numbers of reads per sample have differences due to the methodology, it is impossible to obtain the same number of reads per sample. I mean, with Miseq reads you have relative abundance, you can't talk about total abundance.
Actualiy OTU are carried out intra sample and never inter sample. Within the permissible limits of error you can use the number of reads to compare the abundance of OTU. Lastly I presume that we are supposed to normalize ( I hope you meant standardize) the quantity of DNA before starting PCR.
Thanks for the answer!
If I understood correctly, the number of reads can vary due to methodology independently of the quantity of initial DNA right? This is unfortunate indeed, because I don’t have access to any information about the fly DNA, and therefore I can’t compute any ratio (for example quantity of bacterial dna/quantity of fly dna).
My idea was that standardization to a given quantity of total extracted DNA for all my samples would preserve the bacterial dna/fly dna ratio, and that the following PCR and then MiSeq would also preserve this ratio, allowing to compare samples. Too bad!
Sorry, you can't do a correlation between total DNA extracted and the MiSeq information, because there are for sure bias in DNA extraction, PCR amplification and also the previous mix of DNAs for the MiSeq run. The only thing you can compare is the relative abundance of OTUs between samples, but no total abundance.
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