PNA Clamp Optimisation for Oak Microbiome Studies

Acquiring representative bacterial 16S rRNA gene community profiles in plant microbiome studies can be challenging due to the excessive co-amplification of host chloroplast and mitochondrial rRNA gene sequences that reduce counts of plant-associated bacterial sequences. Peptide Nucleic Acid (PNA) clamps prevent this by blocking PCR primer binding or binding within the amplified region of non-target DNA to stop the function of DNA polymerase. Here, we applied a universal chloroplast (p)PNA clamp and a newly designed mitochondria (m)PNA clamp to minimise host chloroplast and mitochondria amplification in 16S rRNA gene amplicon profiles of leaf, bark and root tissue of two oak species (Quercus robur and Q. petraea). Host chloroplast and mitochondrial 16S rRNA gene sequences were each reduced by averages of 79 %, 46 % and 99 % in leaf, bark and root tissues respectively, leading to an average increase in bacterial reads of 72 %, 35 %, and 17 % in leaf, bark, and root tissue respectively. In leaf and bark where host chloroplast and mitochondria co-amplification was high, inclusion of PNA clamps during PCR resulted in detection of greater bacterial diversity. These results demonstrate that PNA clamps are effective tools for reducing host chloroplast and mitochondria amplification in tree 16S rRNA gene sequencing studies and improving assessments of bacterial community composition.