I am working with low microbial biomass samples which are known to have high contaminating levels of human DNA extracted alongside microbial DNA.
If you determine the DNA concentration after extraction there is a lot of variability between samples. I need to dilute all the samples to ~5 ng/ul prior to the pcr/sequencing steps. However, considering that the DNA concentration includes both microbial and human DNA, will this mess up downstream alpha and beta diversity analyses? (as you are not diluting the microbial DNA consistently between samples?) Or will it not be a problem - as long as you validate any relative taxonomic data with qPCR? I am performing an amplification check now to see if DNA concentration aligns with band brightness after dilution. EDIT: Performed the PCR with the V3-V4 primers on diluted DNA. The very high concentrations post extraction, which were then diluted the most did not have a visible band. This indicates to me that its a higher concentration now from more microbial DNA but from human DNA. I could send a higher concentration away but this leads me back to my original question - does diluting you DNA differently post extraction , prior to the sequencing (indexing etc) and illumina 16S effect your downstream results.