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.

[Vojtěch]

Yes, extremely high (not always, but sometimes). I think its because of PCR producing non-specific products in low mass conditions (https://academic.oup.com/nar/article/24/18/3538/2359394) that highten the number of fragments for sequencing.

But the key question here is if this is still considered to be a negative control, or is it a positive control because of the spike-in?

[GenoMax]

Others with more experimental experience can chime in, but if the negative controls are producing high number of non-specific reads then the experiment will have to be considered as "failed". This is a problem that the experimental people need to address by more rigorous procedures (modeling paper you linked is from 1996 and reagents/instruments have improved significantly since that time). Adding the "spike-in" to negative control, is not solving the underlying problem.

ERCC spike-ins are the most commonly used. While I tried to look for papers, did not see any where they were added to "negative" control.