Fig. 1

Graphical representation of our modified UPDx assay involving nested PCR amplification and two restriction digestion steps and its comparison to earlier UPDx assays. a Conventional PCR with universal primers amplifies primarily host DNA and yields sequencing reads almost entirely derived from the host. b Selective amplification of non-host eukaryote DNA via host-specific restriction enzyme digestion prior to PCR alters the ratio of amplifiable host to parasite DNA, increasing the relative mass of parasite-derived amplicon post-PCR and improving the sensitivity of parasite detection via NGS. c Modification of the assay described in b to a nested approach, facilitating an additional restriction enzyme digestion and additional amplification cycles, reduces the number of host-derived reads and enhances detection of parasite reads via NGS