Evaluation of microbiome enrichment and host DNA depletion in human vaginal samples using Oxford Nanopore’s adaptive sequencing

Metagenomic sequencing is a promising tool for clinical applications to study microbial composition in relation to disease or patient outcome. Alterations of the vaginal microbiome are associated with adverse pregnancy outcomes, like preterm premature rupture of membranes and preterm birth. Methodologically these samples often have to deal with low relative amounts of prokaryotic DNA and high amounts of host DNA, decreasing the overall microbial resolution. Nanopore9s adaptive sampling method offers selective DNA depletion or target enrichment to reject unwanted DNA sequences directly during sequencing without specialized sample preparation. Here, we increase the relative amount of non-human DNA through either direct enrichment or depletion of human DNA. To assess which approach works better, we analyzed 15 clinical routine vaginal swabs with typically high relative amounts of human DNA (> 90%) through metagenomic sequencing. The selective host depletion approach resulted in a 1.70 fold (+/- 0.27 fold) increase in total sequencing depth. Rejected reads contained over 99.80% human reads. In contrast to host depletion, targeted bacterial enrichment resulted in higher human depletion, but the microbial composition was altered as bacterial reads not matching the targets were also depleted. On the other hand, host depletion is not changing the microbial composition, even if high amounts of human host DNA are present, making it a valuable tool for clinical surveillance and medical-based research. However, up to 3% (min: 0.26% max: 2.94%) of human reads were still detectable in the 9accepted9 sequence fraction. Thus, the complete removal of all human host sequences is not yet possible and should be considered as an ethical approval statement might still be necessary.