<p>BLAST results of the unbinned sequences.</p>

Shotgun metagenomics enables simultaneous detection of STHs and microbes in a sample without prior knowledge of what is present. However, validation of shotgun metagenomics with known infection intensity or across different sequencing platforms has not been carried out for eukaryote parasites including STHs, and false positives remain a pervasive issue.

We validated shotgun metagenomics as a method of STH detection in faecal samples. Using the <i>Strongyloides ratti</i> laboratory model of a STH infection we investigated how analytical methods (nucleotide-nucleotide matching, nucleotide-protein matching, marker gene detection, mitochondrial mapping), infection intensity (low and standard laboratory doses) and sequencing technology (short-read vs. long-read) affects sensitivity and specificity of detection. <i>S. ratti</i> was accurately detected at a standard laboratory dose, but low intensity infection were more difficult to detect.