With regard to amplicon-based metagenomics using next generation sequencing, would multiple samples collected from the same well-mixed wastewater treatment basin approximately 10 minutes apart be considered good biological replicates?
I guess the answer depends on the type of system and the resolution your may be looking for. I have never done intraday samples only continuous days. The highest concentration organisms (>2% DNA) have been less variable, where as the lesser fractions seem to be more variable. It probably also depends on the # of reads your doing (>10k).
My gut feeling is to say 10 minutes should be fine considering the average organism is >10 days old. I would be much more concerned about mixing and actually ensuring the sample comes from a homogenous source. Perhaps consider taking it from the RAS instead of the mixed liquor (provided the RAS rate is consistent).
Christopher and Valsa - thanks very much for taking the time to provide suggestions. I'll be doing some pretty deep sequencing (~100k reads/sample). I plan to carry the replicates through sequencing separately and am primarily trying to show that they cluster together and separately from other treatment plants, etc. In other words, to show that differences that I may see between different wastewater treatment plants are significant.
Thanks again for the information.
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