Can a spiked-in negative control still be considered a true negative control in 16S sequencing?

Question

[Vojtěch]

Hi all,

I’m a bioinformatician working on 16S rRNA gene (V3–V4 region) Illumina sequencing data.

In our lab, the ZymoBIOMICS Spike-in Control I (containing Imtechella and Allobacillus) is used as a spike-in for all samples. However, it’s also added to the negative controls.

I’ve been arguing that once the spike-in is added, the “negative control” effectively becomes a positive control, since it now contains known bacterial DNA. The team’s rationale is that if they don’t spike it in, the read counts in the negative control become extremely high or unstable, so they prefer to spike it with the same control as the samples.

Does this approach make sense? And more importantly — can such a spiked sample still be considered a true negative control for contamination assessment?

Any thoughts or references would be greatly appreciated.

[GenoMax]

the read counts in the negative control become extremely high or unstable

Extremely high? That does not seem right. A negative control should have no (or very few reads), assuming the input is just water/buffer.

Looking at the manual for this spike-in it looks like checking the ratio of recovery (besides amount) is important. As long as all samples (including negative control) were treated in the same batch this can be tested in samples (to ensure there is no bias) that do have the spike-in.

If the ratio between the abundance of two including bacteria (I. halotolerans and A. halotolerans) is quite different from the defined value (e.g. 3:7 by 16S copy), this might indicate potential bias in the workflow. For Example, if the abundance of I. halotolerans is much higher than that of A. halotolerans, this might indicate bias during DNA extraction because A. halotolerans is Gram-positive, tougher to lyse than I. halotolerans.