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

Abstract

Abstract Background Current clinical methods for microbial detection in urine are largely culture based and, due to bias and limitations of accuracy and sensitivity, hamper efforts to adequately diagnose and treat urogenital infections. This leads to frequent instances of prolonged and recurrent suffering for women. We report a new method that utilises 3rd generation long-read nanopore sequencing to produce fast, accurate and fully quantitated microbiome profiles appropriate for clinical use that can be immediately utilised to aid diagnosis and focus treatments in cases of recurrent or chronic urinary tract infection, rUTI, cUTI and persistent bacterial vaginosis, BV. 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 able to accurately and reproducibly detect the levels of a mixture of ten species comprising known amounts of hard to lyse gram-positive bacteria, gram-negative bacteria and yeast. Furthermore, we show that, in accordance with previous studies, the female asymptomatic urinary microbiome is largely composed of uromes dominated by Gardnerella vaginitis or one of several Lactobacillus species, L. crispatus, L. iners or L. jensenii. We also confirm the tight relationship between vaginal and urinary populations of the same individual at species and strain levels and provide more evidence for the previously observed dynamic nature of these microbiomes over a menstrual cycle. Conclusions We set out to develop a cost-effective, rapid, unbiased and fully-quantitative microbiome profiling tool appropriate to inform the clinical diagnosis and treatment of common infections. We feel the workflow outlined here can be applied directly to help the numerous women debilitated with urogenital infection, especially chronic or recurrent UTIs and persistent BV, that are served poorly by the current diagnostic systems.