Microbiome analysis using ITS and 16s amplicon sequencing requires specific and efficient primers to amplify the desired regions in complex samples such as plants. It is critical to avoid any plant contamination during these amplification steps. I am wondering if there are any crop specific primers that can be used to amplify fungal and bacterial microbial communities that exist in crops like wheat, barley and oats? Also, which method is best for PCR cleanup; gel extraction or AMPure beads?
Thanks
Florian C Priller
Yes, I am afraid to use just any primers as they would amplify plant based DNA that might overtake the microbial sequences, and would not leave much for further analysis. What do you say?Thanks!
For fungi there are primers are known as ITS1 and ITS4, that amplify the specific region of ITS that contains 5.8s gene, those primers amplify just DNA from fungi because they are specific for fungi, even in basidiomycetes the combination of primers ITS1 and ITS4-B amplify just DNA from this type of fungi. You can use thecniques like RAPD´s or RFLP´s to diferenciate the species involved in microbiomes.
The following articles may clear better yuor doubts:
-ITS primers with enhanced specificity for
basidiomycetes - application to the identification
of mycorrhizae and rusts
M. GARDES and T. D. BRUNS (1993)
-Nuclear ribosomal internal transcribed spacer (ITS)
region as a universal DNA barcode marker for Fungi.
Schoch et. al (2012)
-AMPLIFICATION AND DIRECT
SEQUENCING OF FUNGAL
RIBOSOMAL RNA GENES
FOR PHYLOGENETICS.
White et. al (1990)
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