Does peptide-nucleic acid (PNA) clamping of host plant DNA benefit ITS1 amplicon-based characterization of the fungal endophyte community?

Abstract

Fungal endophyte community amplicon sequencing can lose a significant number of informative reads due to host-plant co-amplification. Blocking of plant-specific sequences with peptide nucleic acid (PNA) clamps has been shown to improve metrics of detected microbial diversity in studies targeting 16S and 18S regions of rRNA genes. However, PNA clamping has not been applied to the plant ITS region of rRNA gene – a widely accepted fungal marker. By applying PNA clamping technique to ITS amplicon sequencing of the endophytic fungal community of elderberry this study shows that PNA clamping significantly reduces host-plant co-amplification with the universal ITS1/ITS4 primer set. However, PNA clamping in combination with the discriminatory ITS1F/ITS2 primer set did not improve the metrics of fungal endophyte community ITS amplicon Illumina sequencing. This study shows that PNA clamping does not add practical benefit to taxonomic profiling of plant-associated fungal communities if the primers are already specific enough to exclude amplification of host DNA. • PNA clamping of host-plant DNA can be applied to fungal marker locus ITS1 . • PNA clamping is beneficial in combination with universal ITS1/ITS4 primer set. • PNA clamping did not improve performance of specific Illumina ITS1F/ITS2 primer set. • Results challenge the practicality of PNA clamping for ITS Illumina sequencing.