Deciding on "negatives" from field-collected samples

[rjoakim]

I have a question about using decontam to determine if the lack of complete sterility during collection is skewing the community compositions of my swab samples.

My field negatives have lots of reads that aren't necessarily exclusive of my study system. Would using them as my negatives be inaffective with this program since they could possibly share strains with my samples at similar abundances?

Additionally, since I sent my template DNA out for library prep and sequencing I only have template concentrations. Would that work as the input concentration data?

I do have extraction negatives, and I added a well with only millipore water, which I consider my library prep negative.

Thank you!

[benjjneb]

My field negatives have lots of reads that aren't necessarily exclusive of my study system. Would using them as my negatives be inaffective with this program since they could possibly share strains with my samples at similar abundances?

decontam was designed in part to detect contaminants using negative controls, without improperly flagging sequences that are shared between real samples and negative controls through processes such as cross-contamination. The key question is this: Is it fair to say that real sequences in your samples should be more likely to be found in samples than in your field controls? That is, it's OK if they might appear in some field controls, but they should appear in a lower fraction of field controls than real samples.

Additionally, since I sent my template DNA out for library prep and sequencing I only have template concentrations. Would that work as the input concentration data?

That should work.

I do have extraction negatives, and I added a well with only millipore water, which I consider my library prep negative.

In general we recommend controls go through at least extraction, since that is a common place in which reagent contamination is added. A single millipore water control might not add much to decontam. Still useful to inspect by hand though.