Is the Juice Worth the Squeeze? Evaluating Peptide Nucleic Acid Blockers for Reducing Plant DNA Contamination in 16S rRNA Gene Sequencing

Next-generation sequencing of microbial communities frequently results in off-target amplification of the host or environmental DNA, which can lead to substantial data loss. This challenge is particularly pronounced in plant-associated microbiome studies, in which commonly used 16S rRNA primers co-amplify mitochondrial and chloroplast DNA. To mitigate this, peptide nucleic acid (PNA) blockers have been applied to reduce amplification of nonbacterial DNA. However, the actual concentrations used vary significantly across studies, and it is not clear whether this affects the retrieved bacterial diversity. In this study, we evaluated the efficacy of universal mitochondrial and chloroplast PNA blockers across a range of concentrations and estimated their impact on alpha and beta diversity. We found that at low concentrations (0.25 µM) PNA blockers efficiently reduced host DNA contamination. Further, they did not have a significant effect on alpha or beta diversity and no bacterial clades appeared to be co-inhibited by the addition of PNA blockers. Although implementing PNA blockers in polymerase chain reaction protocols requires further optimization, our findings demonstrate that the “juice” (enhanced sequencing depth) is indeed “worth the squeeze” (additional effort), as PNA blockers are a powerful tool for improving microbial sequencing outcomes. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .