Enhanced deep metagenomic sequencing of ocular microbes using saponification with the Nanopore platform

Abstract

Purpose : Metagenomic sequencing using Nanopore, allows for the real-time identification of infection-causing pathogens as well as the collective microbiome of the eye. This study investigates the impact of nucleic acid preservation buffers and saponification on microbial DNA recovery, with a primary objective of selectively depleting host DNA and optimizing metagenomic sequencing depth and accuracy.

Methods : Three bacterial species (Enterococcus avium, Klebsiella pneumoniae, Staphylococcus aureus) were spiked with human peripheral blood mononuclear cells (PBMCs) at a 1:10 microbial-to-human cell ratio. Different preservation buffers (DNA/RNA Shield, RNAlater, 95% Ethanol, Liquid Amies, RPMI) were assessed, both with and without saponification. Following incubation in the different buffers, cells were spun down and immersed in phosphate-buffered saline (PBS) with 2.5% saponin to disrupt human cells, allowing for differential lysis via osmotic shock to remove human DNA. Experiments were performed in triplicates, incorporating both positive and negative controls. DNA extraction, followed by whole genome nanopore sequencing was performed using Oxford Nanopore Technologies MinION platform.

Results : Saponification treatment was successful in reducing human DNA observed during sequencing across all preservation buffers. Differential performance was observed across all of the buffers. For Liquid Amies, RNALater and RPMI buffers, the proportion of human reads reduced from 96-98% (without saponification) to 5-9% (with saponification). DNA/RNA Shield showed no K. pneumoniae following saponification due to the gram negative being lysed by the buffer. Ethanol only had a 50% reduction in the total number of human reads. Overall, RNALater and Liquid Amies were effective in removing human reads and preserving all three species of microbes with at least 3-fold depth, and coverage across the entire genome of each species following sequencing.

Conclusions : This study highlights the effectiveness of saponification in depleting host DNA and enhancing microbial enrichment depth when using RNALater and Liquid Amies as preservation buffers. These findings provide practical insights for optimizing experimental protocols, enabling improved microbial DNA recovery in low biomass ocular samples for metagenomic sequencing.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.