A highly accurate nanopore-based sequencing workflow for culture and PCR-free microbial metagenomic profiling of urogenital samples.

Abstract

Background: The application of molecular sequencing methods for microbiome profiling of biological samples are largely restricted to research use. However, they require significant resources such as time and cost and can suffer from amplification biases that may hamper interpretation of complex systems. These issues are also a barrier to adoption as standard clinical tools in, for example, diagnosis of urogenital infections. We report a new method that utilises third generation long-read nanopore sequencing to produce fast, accurate and fully quantitated metagenomic microbiome profiles. Here, as proof of principle, we apply this methodology to reassess the healthy urogenital microbiomes of asymptomatic female and male samples.

Results: We show that our method is capable of accurately and reproducibly detecting both levels and composition of a synthetic mixture of ten species comprising known amounts of hard to lyse gram-positive bacteria, gram-negative bacteria and yeast. When applied to urogenital samples, we confirm previous observations that the female asymptomatic vaginal and urinary microbiomes are predominated by Gardnerella spp. or one of several Lactobacillus species (L. crispatus, L. gasseri, L. iners or L. jensenii) that conform to previously defined community state types. We show the tight relationship between vaginal and urinary populations of the same individual at both species and strain level, provide evidence for the previously observed dynamic nature of these microbiomes over a menstrual cycle and compare biomass and complexity of male and female urobiomes.

Conclusions: We set out to develop an unbiased, amplification and culture-free, fully quantitative metagenomic microbiome profiling tool. Our initial observations suggest our method represents a viable alternative to existing molecular research tools employed in the analysis of complex microbiomes.