Capturing clinically relevant attributes through direct whole genome sequencing of stool.

is the leading bacterial cause of infectious intestinal disease, but the pathogen typically accounts for a very small proportion of the overall stool microbiome in each patient. Diagnosis is even more difficult due to the fastidious nature of in the laboratory setting. This has, in part, driven a change in recent years, from culture-based to rapid PCR-based diagnostic assays which have improved diagnostic detection, whilst creating a knowledge gap in our clinical and epidemiological understanding of genotypes - no isolates to sequence. In this study, direct metagenomic sequencing approaches were used to assess the possibility of replacing genome sequences with metagenome sequences; metagenomic sequencing outputs were used to describe clinically relevant attributes of genotypes. A total of 37 diarrhoeal stool samples with and five samples with an unknown pathogen result were collected and processed with and without filtration, DNA was extracted, and metagenomes were sequenced by short-read sequencing. Culture-based methods were used to validate metagenome-derived genome (MDG) results. Sequence output metrics were assessed for genome quality and accuracy of characterization. Of the 42 samples passing quality checks for analysis, identification of to the genus and species level was dependent on genome read count, coverage and genome completeness. A total of 65% (24/37) of samples were reliably identified to the genus level through MDG, 73% (27/37) by culture and 97% (36/37) by qPCR. The genomes with a genome completeness of over 60% (=21) were all accurately identified at the species level (100%). Of those, 72% (15/21) were identified to sequence types (STs), and 95% (20/21) accurately identified antimicrobial resistance (AMR) gene determinants. Filtration of stool samples enhanced MDG recovery and genome quality metrics compared to the corresponding unfiltered samples, which improved the identification of STs and AMR profiles. The phylogenetic analysis in this study demonstrated the clustering of the metagenome-derived with culture-derived genomes and revealed the reliability of genomes from direct stool sequencing. Furthermore, genome spiking percentages ranging from 0 to 2% total metagenome abundance in the ONT MinION sequencer, configured to adaptive sequencing, exhibited better assembly quality and accurate identification of STs, particularly in the analysis of metagenomes containing 2 and 1% of genomes. Direct sequencing of from stool samples provides clinically relevant and epidemiologically important genomic information without the reliance on cultured genomes.