Design and Validation of a Peptide Nucleic Acid Clamp of Barley and Wheat ITS2 for Fungal Microbiome Surveys

Next-generation sequencing surveys of plant microbiomes often experience considerable loss of reads due to nontarget amplification of host DNA. To alleviate this, peptide nucleic acid (PNA) clamps can be included during PCR amplification to specifically bind and block amplification of host DNA. However, fewer PNA clamps of the plant internal transcribed spacer (ITS) genomic regions exist for fungal microbiome surveys or are available for multi-host comparisons. Here, we designed and validated a novel PNA targeting the ITS2 gene region in barley ( Hordeum vulgare) and wheat ( Triticum aestivum). The PNA reduced nontarget plant amplification by up to 25% in high fungal load samples and did not affect common metrics of microbial diversity (Shannon diversity and community structure). The PNA design pipeline itself is straightforward to implement and requires only freely available software (R, BLASTn, MAFFT); thus, it could be applied to other host systems or gene regions. Development of this PNA will substantially improve the cost efficiency of fungal surveys in two globally important small grain crops, barley and wheat. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2025.