I have and am still extracting (w/ MOBIO PowerSoil kit) DNA from the lettuce rhizosphere in preparation for conducting a microbial analysis of both the fungal and bacterial communities present on and within the lettuce roots. The lettuce is hydroponic so the microbial DNA levels will be low compared to the root cell DNA. I would like to do an unbiased amplification of both 16s (for bacterial community analysis) and ITS (for fungal community analysis) DNA, independently on each sample, for eventual sequencing on the MiSeq.
Does anyone have any opinions on the best 16s and/or ITS primers and/or amplification protocols to fit these needs?
Some additional questions: Should be doing real-time/qPCR on the DNA samples if I wish to do comparisons of the relative levels of different OTUs post-sequencing? My guess is no.
Should I be doing real-time/qPCR on the DNA samples if I wish to do comparisons of the relative levels of different OTUs post-sequencing? My guess is that it is not necessary.
What is the best way to be able to compare relative levels of fungal and bacterial OTU's from amplicons of the same sample (they have to be amplified separately right?) or is this not possible due to variations in the PCR/sequencing?
I have read a few dozen papers where similar techniques are used. There seems to be some differing opinions about which primers to use for the least bias. This obviously changes in the context of the sample. Since the microbiome of hydroponic roots is not well characterized, it makes the selection difficult.
Thus the reason I posted here. I am looking for some opinions and discussion not to be found just reading manuscripts.
here is a good reference for better resolution and accuracy of 16S rDNA based study. "Systematic improvement of amplicon marker gene methods for increased accuracy in microbiome studies" published on Nature biotechnology recently. hope it helps.
http://www.nature.com/nbt/journal/v34/n9/full/nbt.3601.html
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