nextgenusfs
commented
3 years ago Thanks for question @mbcarbonetto. FYI, this was published several years ago here: https://peerj.com/articles/4925/.
In the paper we did essentially two different things: 1) PCR amplified the product from each plasmid separately, cleaned up, quantified, and mixed equally -- this serves as the sequencing control (Stds in some of the heat map figures) and 2) mixed plasmids equally used as template DNA for PCR (we tested this at various concentrations (supplemental).
We did not try to linearize and see if there would be differences there -- I suppose plausible. Since we were focused in this paper on using these data to build a computational pipeline that worked for variable length amplicons (ITS) we largely focused on the PCR/sequencing bias we were seeing in our environmental samples and then of course these biological/synthetic mock samples. But I think the DNA extraction bias is also a very important point -- so we had on our radar to do some spike-in experiments, but effectively those experiments never got done. There is just a lot of variability in microbes, my view essentially is that fungi certainly have a diverse set of properties that are undoubtably going to effect DNA extraction in a mixed community when you consider that aspect and then also consider that PCR is biasing those results further -- it makes it really difficult for me to believe that the read abundance numbers are representative of the underlying fungal community.
mbcarbonetto
Hello, not sure if this is the right place to ask this question, but I will give it a try. As regards the publication i: https://doi.org/10.1101/213470, have you added the SynMock plasmids directly into samples before DNA extraction, or after? have you linearised them? thanks!