Can you easily assess the colonization rate of an inoculated strain (bacteria living in the rhizosphere) by doing a microbiome analysis (NGS of 16S)?
We want to assess the colonization rate of several inoculated bacterial strains(noculated to wheat plants) but we dont have enough time to design strain specific primers (for a qPCR assay). Instead, we want to do a microbiome analysis of the rhizosphere and we wondered if the results given will be good enough to get a quantitative assessment of the strain survival in the rhizosphere. I am afraid that with the microbiome analysis the resolution of taxa identification will not be good enough and that it will be too semi quantitative. We will sequence in advance the target regions (likely the 16S) of the strains to be traced.
Thanks in advance for your answer
Kind regards, Cecile
You're right, with short-read 16S analysis it won't be easy (read: impossible) to distinguish your inoculated strains from indigenous close relatives. I suppose you're not speaking of sterile inoculation tests but about field or pot tests, right?
you have to sequence separately your inoculant strain! you have to maintain a confiend control where no inoculant added! then see the OTUs present in inoculated pot/plant but not in control! possibly those/that OTU belongs to inoculant then see the similarity/clustering by phylogenic tree where your inoculant sequece and identified OTU only present in inoculated pot. Thus you can assess your inoculant success rate individually discriminating from native strain if present in study soil!!
To Joseph Nesme. It will be a pot experiment with natural soil and we intend to get the 16s sequence of the target strain prior to the microbioma analysis. Can it help? Otherwise, how to trace inoculated strains without having to develop strain specific primers? Thanks for your help. Cécile
Sorry for a late reply.
I think that you could always argue that, knowing your sequence rRNA sequence, you'll know which OTU can cluster with it. I'm however quite certain that you won't be able to distinguish your specific strain from closely related OTU. It's for example pretty hard to distinguish based on OTU clustering different strains of Pseudomonas with a different ecology.
It's also quite widely admitted that PCR-based amplicon strategy are considerably skewing "true" community composition (see for ex. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043093 ). As a matter of fact, quantitation is at most semi-quantitative. If you see a terrific difference in abundance from your control, you could probably conclude that you have "more than" and that's it.
Are you considering also a control with a strain known to not be able to colonize roots/rhizosphere ? I'm saying that because in many studies I don't find that control. Residual DNA (even for qPCR) may likely come from already long-time dead bacteria introduced massively during inoculation of millions of bacteria per ml.
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