Suppression/competition PCR: A novel method to minimize unwanted amplicons in metabarcoding, with applications to...

Metabarcoding is widely used for detecting microorganisms in fecal samples, but its effectiveness is often limited by the co-amplification of abundant non-target DNA. In this study, a novel metabarcoding assay was developed to amplify a near-complete 18S rRNA gene fragment suitable for long-read nanopore sequencing, enhancing taxonomic resolution. The primers were optimized to maximize detection of parasitic taxa while minimizing off-target amplification of bacterial and archaeal sequences, thereby improving assay specificity. In this study, the 18S metabarcoding assay worked well on clinical fecal samples containing clinically relevant levels of parasites. However, analysis of ungulate fecal samples revealed that fungal and plant sequences vastly outnumbered other eukaryotic taxa in many samples, obscuring the detection of low-abundance protozoan and helminth parasites. To address this, Suppression/Competition PCR was developed, a novel method that selectively reduces amplification of unwanted DNA. This approach reduced fungal and plant reads by over 99 %, enabling sequences from other taxa to comprise an average of over 98 % of total reads as opposed to an initial 36 %. Utilizing this newly-developed metabarcoding assay in either the standard or Suppression/Competition configuration on fecal DNA extracts from a range of host species, parasites of interest such as Cryptosporidium sp., Cyclospora cayetanensis, Blastocystis sp., Entamoeba sp., Eimeria sp., Ancylostoma sp., and Toxocara sp. were detected, demonstrating its broad applicability.