Samples clustering with negative controls

Hello everyone,

I have a general question regarding negative controls, specifically when you get one sample that clusters together with these controls.

The setup - I've extracted the samples in two batches, each one with its own negative control. Indeed, the sample in question clusters more with the negative control of the respective run but, it is closer to the control than to the remaining samples.

Is there any way to proceed? Should I just remove the sample, since it clusters with the negative controls and not the rest? Or should I "thread carefully" with the negative controls, since they could be cross-contaminated?

I'm also attaching a picture of the plot for the Unweighted Unifrac distance. The blue dots are the controls.

Thanks in advance!

Hi @asbarros,

There are several things to consider.

  • What's the biomass in your samples?
  • What does your plate map look like?
  • Did you do single tube or robotic extraction?
  • Are there any other robotic steps in your preperation?

You might want to look at Quantifying and Understanding Well-to-Well Contamination in Microbiome Research to see if it helps answer some of your questions about what might be happening.


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