I am interested in knowing what kind of quality control (QC) measures we should for NGS and also getting suggestions from microbiome folks on QCs.
We use extraction control (cells of the mock community), sequencing control (DNA of the mock community), negative control (nuclease-free H2O), and internal control (a real sample across all sequencing runs if runs are >1). are these enough or something else we need to consider?
Recently, my colleagues got contamination/reads in the negative control (0.2uM filtered nuclease-free H2O).
What do you suggest here? do another run or use some packages to remove those reads from negative controls and from samples too? if yes, please can you suggest some tools?
I think this is (as with most things) a complex issue. It's going to depend on your type of sample, type of extraction, sequencing platform, etc.
You might find it worth looking at the kathroseq protocol for low biomass samples, as well as a paper on well-to-well contamination in robotic extraction, which might give you a better idea about both controls and possible sources of contamination. Index hopping is a known issue with Illumina sequencing, as well, so you might find this useful.
You might also want to look at the big thread on decontamination on this forum.
Personally, I think I'd replace your nuclease free water with a single organism control, FWIW.
Thanks Justine, this is helpful. I will also into these publications.