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

Metagenomic sequencing is promising for clinical applications to study microbial composition concerning disease or patient outcomes. 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 (> 90%), decreasing the overall microbial resolution. Nanopore's adaptive sampling method offers selective DNA depletion or target enrichment to directly reject or accept DNA molecules during sequencing without specialized sample preparation.

Here, we demonstrate how selective 'human host depletion' resulted in a 1.70 fold (± 0.27 fold) increase in total sequencing depth, thus providing higher taxonomic profiling sensitivity. At the same time, the microbial composition remains consistent with the control experiments. 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. Adaptive sampling increased microbial sequencing yield in all sequenced vaginal samples.

DNA of vaginal swabs was extracted via the ZymoBIOMICS DNA Miniprep kit and sequenced on the GridION (Oxford Nanopore). Adaptive sampling was enabled in the MinKNOW software. In "human depletion experiments", the human reference genome GCA_000001405.28_GRCh38.p13 was used for target depletion.

Our results demonstrated that Oxford Nanopore's unique adaptive sequencing feature has reliably increased the overall sequencing depth of bacterial sequences in clinical metagenomic samples via 'human depletion' without changing the microbial composition during sequencing. Therefore, more samples can be barcoded and sequenced simultaneously, reducing the total cost per sample in a diagnostic laboratory, making molecular monitoring of human reservoirs with low microbial concentrations and high host DNA loads more feasible. We strongly believe that adaptive sampling will prove exceptionally useful within clinical research and the individual microbiological and microbiological diagnostic approach in routine diagnostics.